CN108004213A - A kind of method and kit of CIK cell rapid amplifying - Google Patents

A kind of method and kit of CIK cell rapid amplifying Download PDF

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CN108004213A
CN108004213A CN201810090357.1A CN201810090357A CN108004213A CN 108004213 A CN108004213 A CN 108004213A CN 201810090357 A CN201810090357 A CN 201810090357A CN 108004213 A CN108004213 A CN 108004213A
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段海峰
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Beijing Huizhi Chi Kang Biotechnology Co Ltd
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Abstract

The invention discloses a kind of method and kit of CIK cell rapid amplifying, the technical solution of use is, a kind of kit of CIK cell rapid amplifying, the kit includes activator, CIK cell activator and CIK cell multiplication agent, and the activator is dissolved in PBS buffer preparations by CD3 monoclonal antibodies and IFN γ and forms；CIK cell activator is dissolved in PBS buffer preparations and is formed by IL 2,1 α and IFN γ of IL；CIK cell multiplication agent is made of IL 2, ascorbic acid, vitamin E, albumin human and insulin.The invention has the advantages that：CIK cell rapid amplifying method and kit are easy to use, it is easy to operate, energy is efficient, rapid amplifying CIK cell, greatly shortens cultivation cycle, separated mononuclearcell induced amplification CIK cell in 50mL~100mL peripheral bloods can be reached 2~8 × 10 by two time-of-weeks10It is a.

Description

A kind of method and kit of CIK cell rapid amplifying
Technical field
The present invention relates to the interleaving techniques field of biotechnology, life science and medicine, and in particular to a kind of CIK cell is fast The method and kit of speed amplification.
Background technology
CIK cell, i.e. cytokine induced kill cell (cytokine-induced killer cells), be by The a group foreign cell that human peripheral blood single nucleus cell is induced and obtained jointly with cytokine profiles in vitro.It is thin in LAK Grow up on the basis of born of the same parents (Lymphatic circulation, Lymphokine-activated killer cells) Cytokine activation lymphocyte of new generation.LAK cells are that nineteen eighty-two Grimm etc. is reported first, thin in the single core of peripheral blood Cytokine profiles in vitro culture is added in born of the same parents 4-6 days, a kind of nonspecific killing cell induced, can kill a variety of swollen Oncocyte.Rosenberg seminar first Application IL-2 in 1984 and LAK synergistic treatments 25 clear-cell carcinomas, melanoma, lungs The tumor patients such as cancer, colon cancer, wherein 11 tumor regressions 50%, 1 melanoma disappears completely.The seminar summarizes within 1988 222 tumor patients of synergistic treatment, wherein 16 patient tumors transfer stoves disappear completely, 26 patient tumors disappear 50% More than, the effect of therapy is to metastatic renal cell cancer, melanoma, colon cancer and non_hodgkin lymphoma, is notable.
CD3 in CIK cell+CD56+Double positive cells are its main effects cell, while express two kinds of film eggs of CD3 and CD56 White molecule, itself therefore also known as natural killer cells sample T lymphocytes have the powerful anti-tumor activity of T lymphocytes, thin to tumour The recognition capability of born of the same parents is very strong, killing tumor cell can be accurately identified, without injuring normal cell.Especially opponent is postoperative or putting Patient's significant effect after treatment, can eliminate and remain small metastatic lesion, prevent cancer cell from spreading and recurring, improve immunity of organism Power.China is to carry out the more country of CIK cell clinical test, and relevant person is clinical real by statistics, domestic CIK cell Test and be related to many aspects, for example, the Several Kinds of Malignancy such as liver cancer, stomach cancer, breast cancer, kidney, leukaemia, nasopharyngeal carcinoma, total effective rate For 51.7%.Numerous studies show that CIK cell has than LAK, CTL (cytotoxic T lymphocyte), TIL (tumor-infiltrated lymphs Cell) stronger multiplication capacity and killing activity, it is a kind of new, efficient, the immune effector cell that there is wide spectrum to kill tumor activity, And it is restricted without major histocompatibility complex (MHC), therefore, CIK cell is considered as the tumour adoptive cellular of a new generation The preferred option of immunization therapy, manifests huge application value in immunotherapy of tumors.
At present, the common activation method of CIK cell is that isolated peripheral blood mononuclear cells is used CD3 monoclonals Antibody, interleukin 1 (IL-1 α), interleukin 2 (IL-2) and gamma interferon (English name IFN-γ) etc. because Son is activated, and further long-term cultivation method is expanded by the way of static and interval changes liquid, but generally existing activates Efficiency is low, cultivation cycle is long and the phenomenon of cell growth, cell amplification effect are limited because of nutrients exhaustion or toxic byproduct accumulation Fruit is difficult to meet clinical needs.
The content of the invention
It is an object of the invention to provide a kind of method and kit of CIK cell rapid amplifying, to solve existing CIK Cell amplification efficiency is low, time-consuming, nutrient solution dosage is big and not easy-operating technical problem for culture.
To achieve the above object, technical scheme one is：A kind of method of CIK cell rapid amplifying, including point The step of from peripheral blood mononuclear cells, the method is further comprising the steps of：
1. activation culture bottle：Activator is added into blake bottle to bottom of bottle is covered, is incubated, obtains activation culture bottle, the work Agent is dissolved in PBS buffer preparations by CD3 monoclonal antibodies and IFN-γ and forms；
2. prepare activation medium：The CIK cell activator of 1mL is added into 49mL basal mediums, obtains activation culture Base, the CIK cell activator are dissolved in PBS buffer preparations by IL-2, IL-1 α and IFN-γ and form；
3. it is inoculated with mononuclearcell：Mononuclearcell is resuspended with activation medium, and adds the blood plasma of 10% volume, inoculation In activation culture bottle, 37 DEG C, 5%CO248-96h is cultivated in incubator, obtains CIK cell；
4. expand CIK cell：Activation medium is abandoned, adds amplification culture medium, 37 DEG C, 5%CO2Cultivated in incubator to institute Quantity is needed, the amplification culture medium is the basis training containing IL-2, ascorbic acid, vitamin E, albumin human and insulin Support base.
Preferably, CD3 MAb concentrations are 30-100 μ g/mL in the activator of the step 1., IFN-γ concentration For 1000-3000U/mL；IL-2 concentration is 20000-100000U/mL in the CIK cell activator of the step 2., and IL-1 α are dense Spend for 20000-100000U/mL, the concentration of IFN-γ is 50000-150000U/mL；In the amplification culture medium of the step 4. IL-2 concentration is 500-1000U/L, ascorbic acid concentrations 100-400mg/L, Vitamin E levels 300-800mg/L, people's blood Slurry albumin concentration is 3-8g/L, insulin concentration 5-20ng/L.
It is furthermore preferred that CD3 MAb concentrations are 50 μ g/mL in the activator of the step 1., IFN-γ concentration is 2000U/mL；IL-2 concentration is 50000U/mL in the CIK cell activator of the step 2., and IL-1 α concentration is 50000U/mL, The concentration of IFN-γ is 100000U/mL；IL-2 concentration is dense for 700U/L, ascorbic acid in the amplification culture medium of the step 4. Spend for 200mg/L, Vitamin E levels 500mg/L, albumin human's concentration are 5g/L, insulin concentration 10ng/L.
Preferably, the basal medium is RPMI-1640 basal mediums；3. middle blood plasma is autologous to inactivate for the step Blood plasma, plasma content are the 10% of activation medium volume.
Preferably, 1. middle incubation condition is 4 DEG C~37 DEG C to the step, incubation time 1-18h.
Preferably, when 4. the step expands CIK cell, amplification culture medium was replaced once every 1-3 days.
Technical solution two：A kind of kit of CIK cell rapid amplifying, the kit include activator, CIK cell Activator and CIK cell multiplication agent, the activator PBS buffer preparations are dissolved in by CD3 monoclonal antibodies and IFN-γ and Into；CIK cell activator is dissolved in PBS buffer preparations by IL-2, IL-1 α and IFN-γ and forms；CIK cell multiplication agent by IL-2, ascorbic acid, vitamin E, albumin human and insulin composition.
Preferably, CD3 MAb concentrations are 30-100 μ g/mL in the activator, and IFN-γ concentration is 1000- 3000U/mL；IL-2 concentration is 20000-100000U/mL in CIK cell activator, and IL-1 α concentration is 20000-100000U/ ML, the concentration of IFN-γ are 50000-150000U/mL, and when use dilutes 50 times；IL-2 contents are in CIK cell multiplication agent 500-1000U, ascorbic acid content 100-400mg, content of vitamin E 300-800mg, albumin human's content are 3- 8g and insulin content are 5-20ng, are diluted to 1000mL with basal medium during use.
It is furthermore preferred that CD3 MAb concentrations are 50 μ g/mL in the activator, IFN-γ concentration is 2000U/mL； IL-2 concentration is 50000U/mL in CIK cell activator, and IL-1 α concentration is 50000U/mL, and the concentration of IFN-γ is 100000U/mL, 50 times are diluted during use；In CIK cell multiplication agent IL-2 contents be 700U, ascorbic acid content 200mg, Content of vitamin E is 500mg, albumin human's content is 5g and insulin content is 10ng, with basal medium during use It is diluted to 1000mL.
Preferably, the basal medium is RPMI-1640 basal mediums.
In above-mentioned technical proposal, the method for CIK cell rapid amplifying is first with containing CD3 monoclonal antibodies and IFN-γ Activator coating is incubated blake bottle, and CD3 monoclonal antibodies act on mitogen activity, can be crosslinked, lure with T cell surface C D3 Its activation is led, IFN-γ can induce the synthesis of the factors such as interleukin 1, both coordinate, and compared to being added in culture medium, are The activation of CIK cell provides good substrate, more effectively improves the killing activity of gained CIK cell；Further to contain IL- 2nd, the activation medium of the CIK cell activator and autologous plasma of IL-1 α and IFN-γ stimulates mononuclearcell induced synthesis CIK Cell, wherein, IL-1 α mainly can mediate and raise the expression of peripheral blood lymphocytes surface IL-2R, further also enhance IL-2 promotes lymphocyte growth, propagation and differentiation；On the other hand, cell uses under the excitation of CD3 monoclonal antibodies IL-1 α and IFN-γ, hence it is evident that improve the lethal effect of CIK cell；Finally to contain IL-2, ascorbic acid, vitamin E, people The amplification culture medium culture CIK cell of plasma albumin and insulin, IL-2 can promote propagation and the activation of CIK cell, keep Cell is with higher speed division growth；Needed for albumin human provides primarily as serum substitute, for cell growth Nutrition, in further improved technical solution, using autologous plasma, reduces the incidence of immune response to greatest extent；It is anti-bad Hematic acid and the equal Wheat Protein of vitamin E, vitamin E is fat-soluble, can effectively prevent the oxidation reaction of cell membrane, have The effect of promoting cell Proliferation and activity；Ascorbic acid is the antioxidant of extracellular fluid, and being combined with free radical prevents cell membrane Destroyed be subject to lipid peroxidation, also have the function of to significantly improve DC cells (Dendritic Cells), and then promote T cell to breed, The effect of cytokine secretion.Science adds above-mentioned nutriment, ensure that the efficient fast breeding of CIK cell, especially blood The addition of slurry albumin makes the component of culture medium further make clear, reduces shadow of the allogenic material to CIK cell clinical practice Ring.
The invention has the advantages that：CIK cell rapid amplifying method provided by the invention and kit are easy to use, behaviour Make simply, energy is efficient, rapid amplifying CIK cell, greatly shortens cultivation cycle, improves amplification efficiency；The present invention is being not required In the case of trophoblastic, 7-15 days time can be by separated mononuclearcell induced amplification CIK in 50mL~10mL peripheral bloods Cell reaches 2 × 1010~8 × 1010, it is significantly larger than primary demand amount, clinical practice can be met using a small amount of peripheral blood sample Required CIK cell, hope is provided for clinical CIK cell treatment.
Brief description of the drawings
Fig. 1 is for embodiment 1 with CIK cell obtained by comparative example conventional amplification method to cancer cell killing rate result figure.
Fig. 2 is for embodiment 2 with CIK cell obtained by comparative example conventional amplification method to cancer cell killing rate result figure.
Embodiment
Following embodiments are used to illustrate the present invention, but are not limited to the scope of the present invention, operation involved in embodiment and Method, how specified otherwise, be conventional method；Involved PBS buffer solutions, CD3 monoclonal antibodies, IFN-γ, IL-2 It is conventional reagent unless otherwise specified Deng reagent, cell density is 4 × 105/ mL represents to have in every milliliter of cell suspension 4 × 105A cell.
The method of 1 CIK cell rapid amplifying of embodiment
The present embodiment is used for the method for describing CIK cell rapid amplifying in detail, comprises the following steps：
1. activation culture bottle：Activator is added into blake bottle to bottom of bottle is covered, to 75cm in the present embodiment2Cell culture 10mL activators are added in bottle and are full of bottom of bottle, 4 DEG C of incubation 12h, obtain activation culture bottle, the activator is resisted by CD3 monoclonals Body and IFN-γ are dissolved in PBS buffer solutions (pH7.35~7.45) and are formulated, and CD3 MAb concentrations are 50 μ g/mL, IFN-γ concentration is 2000U/mL；
2. prepare activation medium：5mL autologous plasmas and the CIK cell activation of 1mL are added into 44mL basal mediums Agent, obtains activation medium, matches somebody with somebody wherein the CIK cell activator is dissolved in PBS buffer solutions by IL-2, IL-1 α and IFN-γ System forms, and IL-2 concentration is 50000U/mL, and IL-1 α concentration is 50000U/mL, and the concentration of IFN-γ is 100000U/mL, wherein Autologous plasma is takes anticoagulation cirumferential blood to separate and collect blood plasma after centrifuging 20min with 2000rpm/min, and 56 DEG C inactivate 30min, Obtained by 1500rpm/min centrifugations 10min；
3. separate and be inoculated with mononuclearcell
First, separating peripheral blood mononuclear cells：Anticoagulation cirumferential blood 50~100mL, 2000rpm/min centrifugation 20min is taken, Blood plasma, 56 DEG C of inactivation 30min, and 1500rpm/min centrifugation 10min are separated and collected, autologous plasma is obtained, for preparing activation culture Base；
The sterile PBS dilute bloods of 1~1.5 times of Plasma volumes are separately added into the blood for remove blood plasma, use Ficoll- Paque (is purchased from hundred Aurion of Beijing and wins Science and Technology Ltd., product identification BTN131136) separating peripheral blood mononuclear cells (PBMCs), mononuclearcell is obtained.
Then, mononuclearcell is resuspended in the activation medium 2. prepared with step, and it is 1 × 10 to control inoculum density6A/ ML, is inoculated in 1. activation culture bottle that step is handled, 37 DEG C, 5%CO272h is cultivated in incubator, obtains CIK cell, is inoculated with it Before can gently rinse activation culture bottle 1-2 times with PBS buffer solutions；
4. expand CIK cell：Activation medium is abandoned in centrifugation, is added amplification culture medium, is positioned over 37 DEG C, 5%CO2Incubator Middle culture, is continuing with CIK cell amplification culture medium every 48h and cell density is diluted to 4 × 105/ mL is cultivated, until CIK cell quantity reaches required quantity.The amplification culture medium is to contain IL-2, ascorbic acid, vitamin E, people's blood The basal medium of albumin and insulin is starched, each component concentration is as follows：IL-2 contents 700U/L, ascorbic acid 200mg/L, dimension Raw element E 500mg/L, albumin human 5g/L and insulin 10ng/L.
For the present embodiment after culture 15 days, CIK cell amplification quantity reaches 2 × 1010~8 × 1010It is a, it is far longer than often Advise demand 7 × 109It is a, it is seen then that this method and the reagent energy efficient amplification CIK cell of offer.
For compared with traditional CIK cell amplification method, the present embodiment in the training period, also sets up comparative example, comparative example with Classical culture protocols culture, it is specific as follows：
Anticoagulation cirumferential blood 50~100mL, 2000rpm/min centrifugation 20min is taken, separates and collects blood plasma, 56 DEG C of inactivations 30min, and 1500rpm/min centrifugation 10min, obtain autologous plasma, for preparing activation medium；
The sterile PBS dilute bloods of 1~1.5 times of Plasma volumes are separately added into the blood for remove blood plasma, use Ficoll- Paque separating peripheral blood mononuclear cells (PBMCs), obtain mononuclearcell.
Cell density is adjusted to 1 × 10 using the RPMI1640 culture mediums containing 10% volume hyclone6/ mL, adds Enter IFN-γ, final concentration of 2000U/mL；24 it is small when after add CD3 monoclonal antibodies, the final concentration of 50 μ g/mL of CD3, people is thin in vain Born of the same parents' interleukin -2 (IL-2), the IL-2 final concentration of 50000U/mL of final concentration of 500U/mL, IL-1 α, count every 48h, press later According to 4 × 105The density passage of/mL, while supplement and add IL-2, IL-1 α and IFN-γ.
Contrasted using following (one), (two) two kinds of indexs：
(1) CIK cell proliferation times compare
The CIK cell of above two difference cultural method, respectively amplification the 3rd day of the stage, the 5th day, the 10th day, the 15th It is counted respectively, calculates amplification times, and carry out statistical analysis.As a result it is as follows：
CIK proliferation times compare statistical form
Packet 3d 5d 10d 15d
Comparative example 2.2±0.4 7.5±2.1 98.6±13.9 784.2±43.7
The present invention 3.1±0.3* 12.8±3.4* 164.3±21.7* 1031.5±56.8*
*：It is variant compared with Nostoc commune Vanch method, (P ＜ 0.05)
Upper table is as it can be seen that the amplification method proliferation times in the training period of the present invention will be much higher than Nostoc commune Vanch side Method, has significant difference, on day 3, be within the 5th day, the 10th day, the 15th day 1.41 times of conventional method amplification times respectively, 1.71 times, 1.67 times and 1.32 times.
(2) CIK cell killing activity measures
This cultural method and the cell of classical culture protocols the 9th day phase of amplification are collected, is used afterwards three times with PBS cleaning RPMI1640 nutrient solutions are adjusted to concentration 2 × 10 respectively6The cell suspension of/ml, as effector cell；By exponential phase A549 and K562 cells are made into 2 × 10 respectively5The target cell suspension of/mL；Take respectively the method for the present invention culture effector cell and Each 500 μ L of comparative example effector cell, and 500 μ L target cell suspensions are respectively acting on, mix, as experimental group.To specifications Effector cell's Spontaneous release group (only adding CIK cell 500uL and RPMI1640 nutrient solution 500uL), target cell is set to release naturally Put group (adding target cell 500uL and RPMI1640 culture medium 500uL), target cell maximum release group (only adds target cell 500uL and RPMI1640 culture mediums 500uL and before detection 40min add the Triton-100 of 10 μ L/mL), it is put in incubator Middle culture 6h, gently mixes cell afterwards, and 1000rpm, centrifuges 10min, collects supernatant, and 492nm is measured with lactic dehydrogenase enzyme process The absorbance A of wavelength, each group set 3 multiple holes, and killing activity average value is calculated according to equation below.
As a result shown in Figure 1, the CIK cell obtained by amplification method of the invention and conventional amplification method culture is thin to cancer The killing rate of born of the same parents K562 and A549 are more than 60%, and CIK cell obtained by the method for the present invention is bright to the lethal effect of K562 cells It is aobvious to be higher than conventional method, 70% is quite not less than to A549 killings rate.
The method of 2 CIK cell rapid amplifying of embodiment
The method of the present embodiment CIK cell rapid amplifying is as follows：
1. activation culture bottle：To 75cm213mL activators are added in Tissue Culture Flask to bottom of bottle is covered, 37 DEG C are incubated 1h, Activation culture bottle is obtained, the activator is dissolved in PBS buffer preparations by CD3 monoclonal antibodies and IFN-γ and forms, CD3 Dan Ke Grand antibody concentration is 70 μ g/mL, and IFN-γ concentration is 1500U/mL；
2. prepare activation medium：5mL autologous plasmas and the CIK cell activation of 1mL are added into 44mL basal mediums Agent, obtains activation medium, and the CIK cell activator is dissolved in PBS buffer preparations by IL-2, IL-1 α and IFN-γ and forms, IL-2 concentration is 70000U/mL, and IL-1 α concentration is 70000U/mL, and the concentration of IFN-γ is 80000U/mL, wherein autologous plasma To take anticoagulation cirumferential blood to separate and collect blood plasma, and 56 DEG C of inactivations 30min, 1500rpm/ after centrifuging 20min with 2000rpm/min Obtained by min centrifugations 10min；
3. separate and be inoculated with mononuclearcell
First, separating peripheral blood mononuclear cells：Take anticoagulation cirumferential blood 80ml, 2000rpm/min centrifugation 20min, separation Blood plasma, 56 DEG C of inactivation 30min, and 1500rpm/min centrifugation 10min are collected, autologous plasma is obtained, for preparing activation medium；
The sterile PBS buffer solutions dilute blood of 1 times of Plasma volumes is separately added into the blood for remove blood plasma, is used Ficoll-Paque (be purchased from hundred Aurion of Beijing and win Science and Technology Ltd., product identification BTN131136) separates the single core of peripheral blood Cell (PBMCs), obtains mononuclearcell.
Then, mononuclearcell is resuspended in the activation medium 2. prepared with step, and it is 2 × 10 to control inoculum density6/ mL, It is inoculated in 1. activation culture bottle that step is handled, 37 DEG C, 5%CO272h is cultivated in incubator, CIK cell is obtained, before inoculation Activation culture bottle can be gently rinsed with PBS buffer solutions 1-2 times；
4. expand CIK cell：Activation medium is abandoned in centrifugation, is added amplification culture medium, is positioned over 37 DEG C, 5%CO2Incubator Middle culture, is continuing with CIK cell amplification culture medium every 48h and cell density is diluted to 4 × 105/ mL is cultivated, until CIK cell quantity reaches required quantity.The amplification culture medium is to contain IL-2, ascorbic acid, vitamin E, people's blood The basal medium of albumin and insulin is starched, each component concentration is as follows：IL-2 contents 600U/L, ascorbic acid 300mg/L, dimension Raw element E 400mg/L, albumin human 3g/L and insulin 15ng/L.
For the present embodiment after culture 15 days, CIK cell amplification quantity reaches 7 × 1010It is a.
Similar embodiment 1, in the training period, comparative example is specific as follows with classical culture protocols culture for embodiment：With same Method separation mononuclearcell, unlike, using the RPMI1640 culture mediums containing 10% volume hyclone by cell Density is adjusted to 2 × 106/ mL, adds IFN-γ, final concentration of 1500U/mL；24 it is small when after add CD3 monoclonal antibodies, CD3 Final concentration of 70 μ g/mL, interleukin 2 (IL-2), the IL-2 final concentration of 1400U/mL of final concentration of 600U/mL, IL-1 α, Counted later every 48h, according to 4 × 105The density passage of/mL, while supplement and add IL-2, IL-1 α and IFN-γ.
Contrasted using following (one), (two) two kinds of indexs：
(1) CIK cell proliferation times compare
The CIK cell of above two difference cultural method, respectively amplification the 3rd day of the stage, the 5th day, the 10th day, the 15th It is counted respectively, calculates amplification times, and carry out statistical analysis.As a result it is as follows：
CIK proliferation times compare statistical form
Packet 3d 5d 10d 15d
Comparative example 2.5±0.3 11.4±2.8 129.3±14.4 980.6±42.7
The present invention 3.6±0.2* 16.1±3.1* 193.8±16.1* 1268.4±60.5*
*：It is variant compared with Nostoc commune Vanch method, (P ＜ 0.05)
Upper table is as it can be seen that the amplification method proliferation times in the training period of the present invention will be much higher than Nostoc commune Vanch side Method, has significant difference, on day 3, be within the 5th day, the 10th day, the 15th day 1.44 times of conventional method amplification times respectively, 1.41 times, 1.50 times and 1.29 times.
(2) CIK cell killing activity measures
CIK cell killing activity is measured with method in embodiment 1, and calculates killing activity average value.
As a result shown in Figure 2, the CIK cell obtained by amplification method of the invention and conventional amplification method culture is thin to cancer The killing rate of born of the same parents K562 and A549 are more than 60%, and CIK cell obtained by the method for the present invention is bright to the lethal effect of K562 cells It is aobvious to be higher than conventional method, 70% is quite not less than to A549 killings rate.
Embodiment 3
Exploration based on method, it is following content, dense present invention also offers a kind of kit of CIK cell rapid amplifying Degree is a kind of example, and those skilled in the art can expand concentration and content according to preparation situation and requirement, such as with 10 × (use When 10 times of final concentration), 50 × (during use 50 times of final concentration) etc. are in order to packing, preserve and apply.
The kit includes activator, CIK cell activator and CIK cell multiplication agent.
The activator is dissolved in PBS buffer preparations by CD3 monoclonal antibodies and IFN-γ and forms, and CD3 monoclonals resist Bulk concentration is 30-100 μ g/mL, preferably 50 μ g/mL, and IFN-γ concentration is 1000-3000U/mL, preferably 2000U/mL；
CIK cell activator is dissolved in PBS buffer preparations by IL-2, IL-1 α and IFN-γ and forms；CIK cell activates IL-2 concentration is 20000-100000U/mL in agent, and IL-1 α concentration is 20000-100000U/mL, and the concentration of IFN-γ is 50000-150000U/mL, it is preferred that IL-2 concentration is 50000U/mL, and IL-1 α concentration is 50000U/mL, the concentration of IFN-γ For 100000U/mL, when use, dilutes 50 times；
CIK cell multiplication agent is made of IL-2, ascorbic acid, vitamin E, albumin human and insulin.CIK cell IL-2 contents 500-1000U, ascorbic acid 100-400mg, vitamin E 300-800mg, albumin human 3- in multiplication agent 8g and insulin 5-20ng, preferred content are IL-2 containing 700U, ascorbic acid 200mg, vitamin E 500mg, albumin human 5g and insulin 10ng, is diluted during use, i.e., the final concentration of IL-2 that CIK cell multiplication agent uses with 1000mL basal mediums For 700U/L, ascorbic acid 200mg/L, vitamin E 500mg/L, albumin human 5g/L and insulin 10ng/L.
Embodiment 4
According to the method and step described in embodiment 1, the present embodiment mainly investigates the activation medium of different component, amplification training Amplification efficiency of the base to CIK cell is supported, experiment group is set and processing method is as shown in the table：
Method is tested with reference to described in embodiment 1, unlike, in CIK cell step is expanded, set following real Group and contrast groups are tested, to investigate influence of the amplification culture medium component to amplification efficiency.
*：Being compared with experimental group has significant difference, P ＜ 0.05.
Experiment proves that when each component coordinates in amplification culture medium of the present invention, the facilitation of cell proliferation is most obvious, point About 41.5%, 16.7%, 61.4% and 21.2% are not higher by than other groups.
Embodiment 5
Influence of the present embodiment investigation activation culture bottle to CIK cell phenotype, the method for test method reference embodiment 1, The difference is that experimental group and contrast groups operate as follows in activation culture bottle step：
*：Being compared with experimental group has significant difference, P ＜ 0.05.
Above-mentioned number is it was demonstrated that in the method for the present invention, and activation culture bottle is to main effects cell CD3 in gained CIK cell+ CD56+Double positive cells scale effect is larger, compared to blank control group (comparative example 3) and one pack system control group (1 He of comparative example Comparative example 2), CD3 in experimental group+CD56+Double positive cells ratio substantially increases, even above the sum of three groups of comparative example, illustrates this It is scientific and reasonable to invent two component proportion of activating reagent provided, has cooperative synergism effect, is obviously improved CIK main effects cells CD3+CD56+The ratio of double positive cells.
Although above with general explanation and specific embodiment, the present invention is described in detail, at this On the basis of invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Therefore, These modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to the scope of protection of present invention.

Claims (10)

1. a kind of method of CIK cell rapid amplifying, including the step of separating peripheral blood mononuclear cells, it is characterised in that institute It is further comprising the steps of to state method：
1. activation culture bottle：Activator is added into blake bottle to bottom of bottle is covered, is incubated, obtains activation culture bottle, the activator PBS buffer preparations are dissolved in by CD3 monoclonal antibodies and IFN-γ to form；
2. prepare activation medium：The CIK cell activator of 1mL is added into basal mediums of the 49mL containing blood plasma, must be activated Culture medium, the CIK cell activator are dissolved in PBS buffer preparations by IL-2, IL-1 α and IFN-γ and form；
3. it is inoculated with mononuclearcell：Mononuclearcell is resuspended with activation medium, is inoculated in activation culture bottle, 37 DEG C, 5%CO2Training Support in case and cultivate 48-96h, obtain CIK cell；
4. expand CIK cell：Activation medium is abandoned, adds amplification culture medium, 37 DEG C, 5%CO2Cultivated in incubator to required number Amount, the amplification culture medium are the basis culture containing IL-2, ascorbic acid, vitamin E, albumin human and insulin Base.
2. according to the method described in claim 1, it is characterized in that, CD3 monoclonal antibodies are dense in the activator of the step 1. It is 1000-3000U/mL to spend for 30-100 μ g/mL, IFN-γ concentration；IL-2 concentration in the CIK cell activator of the step 2. For 20000-100000U/mL, IL-1 α concentration is 20000-100000U/mL, and the concentration of IFN-γ is 50000-150000U/ mL；In the amplification culture medium of the step 4. IL-2 concentration be 500-1000U/L, ascorbic acid concentrations 100-400mg/L, dimension Raw element E concentration is 300-800mg/L, albumin human's concentration is 3-8g/L, insulin concentration 5-20ng/L.
3. method according to claim 1 or 2, it is characterised in that CD3 monoclonal antibodies in the activator of the step 1. Concentration is 50 μ g/mL, and IFN-γ concentration is 2000U/mL；IL-2 concentration is in the CIK cell activator of the step 2. 50000U/mL, IL-1 α concentration are 50000U/mL, and the concentration of IFN-γ is 100000U/mL；The amplification cultivation of the step 4. In base IL-2 concentration be 700U/L, ascorbic acid concentrations 200mg/L, Vitamin E levels 500mg/L, albumin human Concentration is 5g/L, insulin concentration 10ng/L.
4. method according to claim 1 or 2, it is characterised in that the basal medium is the culture of RPMI-1640 bases Base；3. middle blood plasma is inactivation autologous plasma to the step, and plasma content is the 10% of activation medium volume.
5. method according to claim 1 or 2, it is characterised in that 1. middle incubation condition is 4 DEG C~37 DEG C to the step, Incubation time is 1-18h.
6. method according to claim 1 or 2, it is characterised in that when the step expands CIK cell 4., amplification cultivation Base was replaced once every 1-3 days.
7. a kind of kit of CIK cell rapid amplifying, it is characterised in that the kit includes activator, CIK cell swashs Agent living and CIK cell multiplication agent, the activator are dissolved in PBS buffer preparations by CD3 monoclonal antibodies and IFN-γ and form； CIK cell activator is dissolved in PBS buffer preparations by IL-2, IL-1 α and IFN-γ and forms；CIK cell multiplication agent by IL-2, Ascorbic acid, vitamin E, albumin human and insulin composition.
8. kit according to claim 7, it is characterised in that CD3 MAb concentrations are 30- in the activator 100 μ g/mL, IFN-γ concentration are 1000-3000U/mL；IL-2 concentration is 20000-100000U/mL in CIK cell activator, IL-1 α concentration is 20000-100000U/mL, and the concentration of IFN-γ is 50000-150000U/mL, and when use dilutes 50 times；CIK In cell expansion agents IL-2 contents be 500-1000U, ascorbic acid content 100-400mg, content of vitamin E 300- 800mg, albumin human's content are 3-8g and insulin content is 5-20ng, and when use is diluted to basal medium 1000mL。
9. kit according to claim 7, it is characterised in that CD3 MAb concentrations are 50 μ in the activator G/mL, IFN-γ concentration are 2000U/mL；IL-2 concentration is 50000U/mL in CIK cell activator, and IL-1 α concentration is 50000U/mL, the concentration of IFN-γ are 100000U/mL, and when use dilutes 50 times；IL-2 contents are in CIK cell multiplication agent 700U, ascorbic acid content 200mg, content of vitamin E 500mg, albumin human's content are 5g and insulin content For 10ng, 1000mL is diluted to basal medium during use.
10. the kit according to claim 7 or 8, it is characterised in that the basal medium is RPMI-1640 bases Culture medium.
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