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CN103756963B - A kind of method of amplification in vitro NK cell - Google Patents

️Tue Oct 29 2019

CN103756963B - A kind of method of amplification in vitro NK cell - Google Patents

A kind of method of amplification in vitro NK cell Download PDF

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Publication number

CN103756963B

CN103756963B CN201310684685.1A CN201310684685A CN103756963B CN 103756963 B CN103756963 B CN 103756963B CN 201310684685 A CN201310684685 A CN 201310684685A CN 103756963 B CN103756963 B CN 103756963B Authority

China

Prior art keywords

cell

amplification

antibody

culture

added

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2012-12-13

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CN201310684685.1A

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Chinese (zh)

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CN103756963A (en

Inventor

叶永清

柳运猛

杨雪晶

苏国新

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Shanghai Ke Laixun Bioisystech Co Ltd

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Shanghai Ke Laixun Bioisystech Co Ltd

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2012-12-13

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2013-12-13

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2019-10-29

2013-12-13 Application filed by Shanghai Ke Laixun Bioisystech Co Ltd filed Critical Shanghai Ke Laixun Bioisystech Co Ltd

2013-12-13 Priority to CN201310684685.1A priority Critical patent/CN103756963B/en

2014-04-30 Publication of CN103756963A publication Critical patent/CN103756963A/en

2019-10-29 Application granted granted Critical

2019-10-29 Publication of CN103756963B publication Critical patent/CN103756963B/en

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2033-12-13 Anticipated expiration legal-status Critical

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Abstract

The present invention relates to a kind of culture amplification method of NK cell, specially a kind of method for cultivating massive amplification NK cell in vitro with autologous peripheral blood mononuclear cell, this method achievees the purpose that proliferation and activated NK using different combination of cytokines.Autologous peripheral blood mononuclear cell is specially seeded in the pre-coated culture bottle moderate stimulation 16 of AntiCD3 McAb, CD16 antibody~for 24 hours, the serum-free medium culture containing IL-2 and IL-18 is added.The present invention solidifies AntiCD3 McAb, CD16 antibody coating, directly stimulate activated NK, significantly improve the killing toxicity of NK cell, only activation and amplification to NK cell are achieved that by two kinds of cell factors of IL-2 and IL-18, not only the amplification times and cytotoxicity of NK cell ensure that, but also greatly reduced the toxigenic capacity of NK cell.

Description

A kind of method of amplification in vitro NK cell

[technical field]

The present invention relates to a kind of method for cultivating massive amplification NK cell in vitro with autologous peripheral blood mononuclear cell, tools Body is related to a kind of achieving the purpose that proliferation and activated NK using the combination of cytokines and processing mode of various concentration.

[background technique]

Natural killer cells (natural killer cell, NK) is to belong to lymphocyte linage, contains perforin and grain The cellulotoxic lymphocyte of enzyme granulate.It is restricted without MHC to the identification of target cell, without presensitization can direct killing it is swollen Oncocyte, can also secrete cytokines adjust the functions of other immunocytes, be the main undertaker of the body innate immunity, and The core of acquired cellular resistance adjusts cell, plays important work in tumour immunity, viral infection resisting and the non-own cell of removing With.There are some researches prove natural killer cells to be not only the important function ingredient in natural immune system in fact, it, which equally has, obtains Obtain some features of property immunocyte.In immune system, the speed of NK cell effect is faster than T cell or B cell, effect Mechanism mainly identifies target cell, by release perforin, granzyme and secretion cytokine profiles, dissolves certain tumours rapidly Cell plays the effect for adjusting immune and hemoposieis and direct killing target cell.But since NK cell is in human peripheral Content is extremely low and tumor tissues in distribution frequency it is lower (< 10%), only account for the 5%-10% of mononuclearcell, greatly limit The application of NK cell clinically as adoptive immunity cell is made.For many years, people always try to realize that in vitro NK is thin The massive amplification of born of the same parents, but current routine techniques be mainly added in cultivating system in vitro IL-2, IL-12, IL-15 and The combinations of factors such as γ-IFN promote the amplification of NK, only NK cell can be made to expand several times or ten after culture after a period of time Several times, purity is also undesirable, therefore can not meet actual application.And this method factor demand is larger, cost compared with Height, culture effect is unstable, is not suitable for external large-scale application.

[summary of the invention]

The problems such as in order to solve undesirable purity after NK cell expands in the prior art, higher cost, the present invention provides one The method of the amplification in vitro of kind new NK cell, can obtain that a large amount of, application is safe, purity is higher under condition of culture in vitro And NK cell that killing activity is strong so that the cell reaches and can safely and effectively be applied to clinical quality and refer to Mark.

To achieve the above object, a kind of method that the present invention designs amplification in vitro NK cell, it is characterised in that by following step It is rapid to constitute:

A. the CD16 monoclonal of CD3 monoclonal antibody, 0.01~5.0mg/ml that concentration is 0.01~5.0mg/ml is resisted Body is coated in Tissue Culture Flask, and coating condition is room temperature, and incubation time is 0.5~12h；

B. the mononuclearcell obtained will be separated from peripheral blood, and above-mentioned cell culture is seeded in serum-free medium In bottle, stimulation 2~for 24 hours；

C. NK cell culture serum-free medium and concentration is added as the interleukin 2 and 15 of 15~50ng/ml The interleukin-18 of~50ng/ml stimulates 12~72h, is then transferred to and continues to cultivate in cell culture bags；

D. NK cell is harvested.

The present invention is from clinical practice application feasibility, under the premise of considering safety, validity, by cell Source, the type of culture medium of application, combination of cytokines and NK cell clinically practical application when patient's acceptable cost etc. Various aspects are comprehensive to be measured, and optimization optimum N K cell culture expands scheme, is exempted to adopt in clinic to can be realized NK cell The large-scale application of epidemic disease therapy improves clinical efficacy and mitigates patient economy burden and lay the foundation.

The present invention is using AntiCD3 McAb, the formal layout of the preparatory coated cell culture bottle of CD16 antibody, and initiator cell is using self Peripheral blood mononuclear cells, concentration are preferably 1.0 × 10⁶/ ml cultivates thorn in the culture bottle after AntiCD3 McAb, CD16 antibody coating Sharp 16~for 24 hours, the use concentration of the IL-2 and IL-18 are preferably 15ng/ml.

Concentration of the anti-cd 3 antibodies in coating buffer is 0.15mg/ml.

Concentration of the anti-CD16 antibody in coating buffer is 0.15mg/ml.

By the cell training after separating the mononuclearcell addition AntiCD3 McAb obtained, CD16 antibody coating in autologous peripheral blood Support bottle moderate stimulation 16h.

Interleukin 2 concentration used is 15ng/ml in medium exchange combination.

Interleukin-18 concentration used is 15ng/ml in medium exchange combination.

Compared with the conventional NK cell culture processes for only adding cytokine profiles into cultivating system merely, the present invention It has the advantages that

1. coated AntiCD3 McAb, CD16 antibody directly can quickly and effectively make NK cell by signal stimulus, activate NK Cell promotes the synthesis of cell factor (TNF, IFN-γ etc.), significantly improves the killing toxicity of NK cell.

2. the processing of turn bag: after being cultivated 3~5 days in coated culture bottle, then cell being transferred in cell culture bags, in this way Both it can guarantee that NK cell was sufficiently activated, and in turn avoid the apoptosis that high concentration antibody induces the continuous action of cell, and The effect of cell culture bags is better than Tissue Culture Flask under equal conditions；

3. being achieved that activation and amplification to NK cell only by two kinds of cell factors of IL-2 and IL-18, IL-2 can NK cell is stimulated to express a large amount of IL-2R α chain, to make NK cell largely be proliferated, and the expression of NK cell is glutinous under IL2 stimulation Attached molecule increases the particle in NK cytoplasm and promotes the expression of serine easterase mRNA, to improve the cell toxicant of NK cell Activity；And IL-18 can be with induced NK cell secretion of gamma-IFN, and has killing for the proliferation for promoting NK cell, activation and Fas mediation Hurt function, and can have the function that promote NK cell activation by ITAM intracellular.It is used in conventional NK cell culture system Combinations of factors in also used IL-12, IL-15 and IFN-γ etc., the present invention has only used two kinds in cultivating system in vitro Cell factor, not only ensure that the amplification times and cytotoxicity of NK cell, but also reduce the cost of cell culture.

[Detailed description of the invention]

Fig. 1 is present invention figure compared with conventional culture methods NK cells expanded；

Fig. 2 is NK cell purity figure obtained by the method for the present invention；

Fig. 3 is NK cell purity figure obtained by conventional culture methods；

Fig. 4 is present invention figure compared with conventional culture methods gained NK cell is to K562 cell killing activity.

[specific embodiment]

In order to make the objectives, technical solutions, and advantages of the present invention clearer, the present invention is carried out further detailed Explanation.Production equipment in the application is all the commonly used equipment of this field, it should be understood that embodiment what follows is only to example Show the present invention, is not intended to limit the present invention.Unless otherwise specified, method therefor is conventional method in embodiment, instrument used Device, material, reagent etc. can be obtained through commercial channels unless otherwise specified.

Embodiment 1

One, the separation of peripheral blood mononuclear cells (PBMC) and the culture of NK cell

The coating buffer containing AntiCD3 McAb, CD16 antibody is added in Tissue Culture Flask in advance, is incubated at room temperature 1h, antibody is dense eventually Degree is preferably 0.15mg/ml.

Peripheral blood mononuclear cells (PBMC) is acquired by blood cell separator, the blood sample of acquisition is gone into centrifugation Pipe；700g is centrifugated 10min, draws spare when upper plasma culture；Sample is restored to 0.9% physiological saline blood sample Original volume mixes；Diluted blood is slowly added on Ficoll, 900g, is centrifuged 20min；It is single to draw separating liquid interface milky Nucleus layer；Centrifuge washing 2 times simultaneously counts；PBMC is resuspended with serum free medium, and adjusts cell concentration to 1.0 × 10⁶/ ml。

It is transferred in advance with after AntiCD3 McAb, the coated Tissue Culture Flask moderate stimulation 16h of CD16 antibody, the IL-2 of 15ng/ml is added Continue to cultivate 72h with the IL-18 of 15ng/ml.

It hereafter is 1 × 10 with the serum free medium adjustment cell concentration containing 0.5% human serum albumin⁶/ ml, is added IL-2 and IL-18 is transferred in cell culture bags and cultivates, add within every 2-3 days the nothing of IL-2 containing same concentrations and IL-18 to original content Blood serum medium maintains cell concentration 2 × 10⁶High-purity, the high toxicity of massive amplification can be obtained in/ml or so after 14 days NK cell.

Two, this method is compared with the cells expanded of conventional method

Culture cell was taken from two groups at the 0th, 7,14 and 21 day respectively, with counting after Trypan Blue, the same day will be counted For total number of cells divided by the cell number (i.e. the 0th day) before culture, numerical value is the amplification times of cell.It in this approach can dynamic ratio Compared with the amplification situation of two groups of cells, as a result as shown in table 1 and Fig. 1: at the 7th, 14,21 day of culture, the NK cell of this law was expanded Multiple is all remarkably higher than conventional group P < 0.01.

Table 1:

Note: " * * " refers to for conventional group, p < 0.01

Three, this method is compared with the cell purity of conventional method

Cell culture fluid was taken from two groups of cultivating systems respectively at the 21st day, PBS is washed twice, and cell concentration is adjusted after washing It is 2 × 10⁵Streaming antibody is added in/ml, and 4 DEG C are protected from light 30min, washes off Excess antibody, and the streaming for carrying out cell phenotype is resuspended in PBS Detection.Antibody used comes from U.S. company BD, as a result, it has been found that the CD3 of conventional group (Fig. 3)CD(16+56)⁺NK cell accounts for 81.27 ± 1.12%, this method (Fig. 2) CD3CD(16+56)⁺NK cell accounts for 91.93 ± 1.91%, two groups there was no significant difference (P > 0.05)。

Four, this method is compared with conventional method gained NK cell is to K562 cell killing activity

Using the K562 cell in logarithmic growth phase as target cell, it is adjusted to 1 × 10⁵/ mL, two methods are trained 21 days NK cells are supported as effector cell, mix effector cell and target cell by the effect target ratio of 1: 1 and 2: 1 and 5: 1 and 10:1, Laying effect cell hole, Target cell wells simultaneously, every group is all provided with 3 parallel holes, and every hole final volume is 200 μ l, 37 DEG C, 5%, CO₂Training It supporting and is incubated in case, 20 μ l CCK-8 solution are added in every hole after 12h, continue to be incubated for 4h, OD value when 450nm is detected with microplate reader, Killing rate is calculated as follows:

Killing rate (%)=[1- (experimental port OD value-effect hole OD value/Target cell wells OD value)] × 100%

As a result as shown in table 2 and Fig. 4, in different effect target ratios, killing activity of this method gained NK cell to K562 cell It is all remarkably higher than conventional group (P < 0.01).

Table 2:

Note: " * * " refers to for conventional group, p < 0.01

Claims (1)

1. a kind of method of amplification in vitro NK cell, it is characterised in that be made of following steps:

The coating buffer containing AntiCD3 McAb, CD16 antibody is added in Tissue Culture Flask in advance, is incubated at room temperature 1h, antibody is final concentration of 0.15mg/m,

Peripheral blood mononuclear cells are acquired by blood cell separator, the blood sample of acquisition is gone into centrifuge tube；700g, centrifugation 10min is separated, is drawn spare when upper plasma culture；Sample is restored to original volume with 0.9% physiological saline blood sample, is mixed；It will Diluted blood is slowly added on Ficoll, 900g, is centrifuged 20min；Draw separating liquid interface milky mononuclearcell layer；Centrifugation is washed It washs 2 times and counts；PBMC is resuspended with serum free medium, and adjusts cell concentration to 1.0 × 10⁶/ ml,

Be transferred to the IL-2 that 15ng/ml with AntiCD3 McAb, after the coated Tissue Culture Flask moderate stimulation 16h of CD16 antibody, is added in advance and The IL-18 of 15ng/ml continues to cultivate 72h,

It hereafter is 1 × 10 with the serum free medium adjustment cell concentration containing 0.5% human serum albumin⁶/ ml, add IL-2 and IL-18 is transferred in cell culture bags and cultivates, the serum-free of every 2-3 days addition IL-2 containing same concentrations and IL-18 is trained to original content Base is supported, maintains cell concentration 2 × 10⁶The NK cell of amplification can be obtained in/ml after 14 days.

CN201310684685.1A 2012-12-13 2013-12-13 A kind of method of amplification in vitro NK cell Active CN103756963B (en)

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Application Number	Priority Date	Filing Date	Title
CN201310684685.1A CN103756963B (en)	2012-12-13	2013-12-13	A kind of method of amplification in vitro NK cell

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CN2012105416584		2012-12-13
CN201210541658.4		2012-12-13
CN2012105416584A CN102994449A (en)	2012-12-13	2012-12-13	Method for in-vitro amplification of NK cells
CN201310684685.1A CN103756963B (en)	2012-12-13	2013-12-13	A kind of method of amplification in vitro NK cell

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CN103756963A CN103756963A (en)	2014-04-30
CN103756963B true CN103756963B (en)	2019-10-29

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