CN106350488B - PD-1 for treating tumour closes the preparation method of CIK - Google Patents

PD-1 for treating tumour closes the preparation method of CIK Download PDF

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CN106350488B
CN106350488B CN201610833367.0A CN201610833367A CN106350488B CN 106350488 B CN106350488 B CN 106350488B CN 201610833367 A CN201610833367 A CN 201610833367A CN 106350488 B CN106350488 B CN 106350488B
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cell
culture
monoclonal antibody
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suspension
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CN106350488A (en
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袁小林
杨真
袁儒非
孙秀艳
李纯
崔益芬
关庆琳
张青
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Beijing Jiamei Kanglian Medical Technology Co ltd
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Dalian University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2302—Interleukin-2 (IL-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/24—Interferons [IFN]

Abstract

PD-1 for treating tumour closes the preparation method of CIK, belongs to medical field, anti-human CD3 monoclonal antibody coated cell culture bottle；Step is activated, activates culture lymphocyte using the CD3 monoclonal antibody, IFN γ and IL-2；Cleaning step, cleaning activate the lymphocyte after culture three times；Step is closed, the lymphocyte after the cleaning that suspended with physiological saline obtains suspension cell solution, PD-1 monoclonal antibody is added in Xiang Suoshu suspension cell solution, is suspended and is incubated for；Cell suspension after being incubated for the PD-1 monoclonal antibody adds physiological saline and feedback cell suspension is made.The method of the present invention, which prepares the closed CIK of PD-1, has the effect of the features such as dosage of PD-1 monoclonal antibody is few, closing PD-1 molecule is good, anti-tumor activity is high.It is provided simultaneously with the characteristics of easy to operate, preparation cost is low and is convenient for clinical application.

Description

PD-1 for treating tumour closes the preparation method of CIK
Technical field
The present invention relates to medical fields, especially for treating the preparation method of the PD-1 closing CIK of tumour.
Background technique
Programmed death receptor 1 (PD-1) is a kind of protein molecule for being expressed in T cell surface, as PD-1 and its ligand PD-L1 or PD-L2 can inhibit the transcription of downstream NF- κ 1 B gene when combining, inhibit the secretion of interferon-δ, to inhibit T cell Killing activity.PD-L1 or PD-L2 are expressed in kinds of tumor cells, such as lung cancer, breast cancer, gastric cancer, the cancer of the esophagus, hepatocellular carcinoma, evil Property the tumor tissues height such as melanoma, oophoroma, cancer of pancreas, clear-cell carcinoma and bladder transitional cell carcinoma express PD-L1.T cell table The T cell cytotoxic activity inhibition PD-1 in face caused in conjunction with the PD-L1 being expressed on tumour cell is that tumour cell is escaped Keep away one of the important mechanisms of immunosurveillance and killing.Using PD-1 antibody or PD-L1 antibody blocking PD-1 and PD-L1, PD-L2 In conjunction with the tumor activity that kills of T cell being made to be not inhibited, to improve the effect that T cell kills tumor.Research confirms to use PD-1 Dan Ke The tumours such as grand antibody venoclysis treatment malignant mela noma, lung squamous cancer, colon cancer and kidney receive good therapeutic effect.So And venoclysis enters intracorporal PD-1 antibody for by the hemodilution of human body 4000ml-5000ml, therefore in order to remain effective The amount that drug concentration needs to be transfused PD-1 antibody is larger.Blood constituent, lymphoid tissue microenvironment structure and composition are complicated simultaneously, no Only will cause PD-1 antibody loss also influence whether PD-1 antibody and T cell surface PD-1 combination.
CIK is the killing cell of cytokine profiles induction, and existing CIK activation cultural method is easily obtained a large amount of effect Cell.However these effector cells have higher PD-1 expression rate, this affects the tumor-killing effect of CIK to a certain extent And clinical efficacy.Applying the stage patented technology CN201511018960.1, CN201610278810.2, PD-1 is added during autologous peripheral blood culture in vitro of lymphocytes in CN201410573897.7 and CN201410573311.7 Antibody is washed when collecting cell and abandons unbonded PD-1 antibody.Treatment is usually existed with training base unit weight in lymphocyte incubation 1500ml or so, the total volume for training base is larger, and the dosage of PD-1 antibody is still larger, while the PD-1 antibody being not associated with after cultivating It is discarded washing cell Shi Suipei base, causes certain waste.In addition, the secretion and metabolite of cell during the cultivation process, The combination of the PD-1 antibody and PD-1 that can influence to a certain extent, to influence the sealing effect of PD-1.
Treat expensive, addition PD-1 antibody, the PD-1 antibody in venoclysis or CIK incubation with PD-1 antibody The drawbacks such as big, the at high cost and PD-1 sealing effect susceptible of dosage, greatly affected the clinical application of PD-1 antibody.
Summary of the invention
It is an object of the invention to overcome above-mentioned the deficiencies in the prior art, it is desirable to provide a kind of cell Proliferation multiple is big, kills The preparation method for the Tumor cytotoxicity cell that wound activity is high and preparation cost is low.The technical solution of the present invention is as follows: for treating The preparation method of the PD-1 closing CIK of tumour, comprises the following steps: anti-human CD3 monoclonal antibody coated cell culture bottle；Activation Step activates culture lymphocyte using the CD3 monoclonal antibody, IFN γ and IL-2；Cleaning step, cleaning swash three times The lymphocyte after culture living；Step is closed, the lymphocyte after the cleaning that suspended with 20-50ml physiological saline obtains To suspension cell solution, PD-1 monoclonal antibody is added in Xiang Suoshu suspension cell solution, 18-25 DEG C of suspension is incubated for 30 minutes must To cell suspension.
Preferably, the preparation method of the PD-1 closing CIK for treating tumour further includes step: filtering step Suddenly, it is added in Xiang Suoshu cell suspension after the filtering of 200 mesh filter screen of cell-dispersing agent and adds physiological saline and fed back to 300ml Liquid.
Preferably, the anti-human CD3 monoclonal antibody coated cell bottle comprises the following steps: using 0.01M PH9.6's PBS solution dilutes first class and purifies anti-human CD3 monoclonal antibody to concentration 50-70ng/ml；First class purifying after dilution is anti- People's CD3 monoclonal antibody is added in culture bottle, and 4 DEG C are placed 24 hours or more.
Preferably, the activation step comprises the following steps: by heparin anti-coagulating inject centrifuge tube A in, at a temperature of 4 DEG C with 3000rpm is centrifuged 5min；Upper plasma is taken to move into centrifuge tube B, 56 DEG C of water-bath 30min are centrifuged at a temperature of 4 DEG C with 3000rpm 15min collects supernatant, and 4 DEG C save backup；Lower layer's haemocyte stays spare in centrifuge tube A；The physiological saline suspension centrifuge tube The haemocyte in A, it is 1.077 ± 0.001 that blood cell suspension obtained, which is slowly added into the density equipped with 4ml, In the centrifuge tube of Ficoll-Hypaque, 20min is centrifuged with 3000rpm at a temperature of 4 DEG C；Collect mononuclearcell confluent monolayer cells simultaneously The cell is cleaned three times using centrifugal process；Anteserum-less substrate is added to be resuspended, anteserum-less substrate suspension cell is made；It will be described It is added in anteserum-less substrate suspension cell containing in the anti-human coated culture bottle of CD3 monoclonal antibody, is added and inactivates blood It starches and adds into anteserum-less substrate, 37 DEG C, 5%CO2Under the conditions of cultivate 24 hours；IFN γ, 37 DEG C, 5% are added into culture bottle CO2Under the conditions of cultivate 4-6 days；Take culture after cell move into cell culture bags in, be added anteserum-less substrate, inactivation blood plasma and IL-2,37 DEG C, 5%CO2Under the conditions of cultivate 4-5 days；Add anteserum-less substrate and IL-2,37 DEG C, 5%CO2Under the conditions of cultivate 4-5 It.
Preferably, also include before the cleaning step: checking procedure detects cell quantity after carrying out cell culture, and Utilize the mycoplasma in round pcr detection culture supernatant.
The invention has the benefit that the dosage of PD-1 monoclonal antibody is few, PD-1 molecule sealing effect is good, PD-1 closing CIK anti-tumor activity it is high.Have the characteristics that easy to operate, preparation cost is low and is convenient for clinical application simultaneously.
Detailed description of the invention
Fig. 1 is the expression rate of the PD-1 molecule of CIK cell；
Fig. 2 is the recall rate that PD-1 monoclonal antibody closes CIK cell PD-1 molecule；
Fig. 3 is compared with CIK closes the tumor cell destruction of CIK with PD-1.
Specific embodiment
With reference to the accompanying drawing, by specific embodiment, the invention will be further described.Following embodiment is descriptive , it is not restrictive, this does not limit the scope of protection of the present invention.
Experiment equipment
Low temperature normal speed centrifuge (U.S., Beckman), CO2Cell incubator (Japan, Sanyo), pipettor (U.S., Thermo), measuring pipette (U.S., Kimble), 75cm2Culture bottle (U.S., Corning), 15ml centrifuge tube (U.S., Corning), 50ml centrifuge tube (U.S., Corning), cell scraper (U.S., Corning), cellular filter (U.S., Corning), inverted microscope (Japan, Nikon), cryopreservation tube (U.S., Corning), 96 porocyte culture plates (U.S., Corning), microplate reader (U.S., BioTek), flow cytometer (U.S., BD).
Experiment reagent
Ficoll-Hypaque cell separating liquid (the Tianjin ocean Hao biological products science and technology limited Company)), sodium chloride note Penetrate liquid (four medicine company of Shijiazhuang), heparin sodium (Shanghai No.1 Bio-Chemical Pharmacetical Industry Co., Ltd), IFN-γ (Beijing Fourth Ring bio-pharmaceuticals Co., Ltd), IL-2 (Beijing Shuanglu Pharmaceutical Co., Ltd.), first class purify anti-human CD3 monoclonal antibody (U.S., EBioscience), anteserum-less substrate (U.S., Thermo), human serum albumins (the limited public affairs of Shanxi health treasured biological products share Department), Pembrolizumab (U.S., MSD), FITC label the anti-human CD3 monoclonal antibody of mouse (U.S., BD), PE mark rabbit-anti people PD-1 monoclonal antibody (U.S., eBioscience), Hank ' s liquid (preparation method: 1. solution As: NaCl 160g, KCl8g, MgSO4·7H2O 2g、MgCl2·6H2O 2g is added to 800ml tri-distilled water, dissolution.CaCl22.8g is dissolved in 100ml tri-distilled water, molten It is added to after solution in above-mentioned 800ml solution, supplies tri-distilled water to 1000ml.2. second liquid: Na2HPO4·12H2O 3.04g、KH2PO4 800ml distilled water, dissolution is added in 1.2g, glucose 20g.0.4% phenol red solution 100ml is added, above-mentioned two liquid mixing adds three steamings Water is to 1000ml.By 1 part of solution A, 1 part of second liquid, 18 parts of tri-distilled water filtrations when use, 8 pounds sterilize for 20min minutes, 4 DEG C of preservations), NaCl (examination of traditional Chinese medicines Lu), KCl (examination of traditional Chinese medicines Lu), MgSO4·7H2O (Tianjin Zhi Yuan Chemical Co., Ltd.), MgCl2·6H2(the day O Jin Zhiyuan Chemical Co., Ltd.), CaCl2(Tianjin Bo Di Chemical Co., Ltd.), Na2HPO4·12H2O (Tianjin recovery science and technology Development Co., Ltd), KH2PO4(Tianjin Zhi Yuan Chemical Co., Ltd.), glucose (examination of traditional Chinese medicines Lu), phenol red (the upper graceful biology of Hypon Science and Technology Ltd.), SCG7901 stomach cancer cell (Shanghai cyropreservation center).
CD3 monoclonal antibody coating: it is anti-that anti-human CD3 monoclonal is purified using the PBS solution dilution first class of 0.01M PH9.6 Body is added to 75cm to concentration 50-70ng/ml, by this anti-human CD3 monoclonal antibody2In culture bottle, every bottle of 30-50ml, 4 DEG C are put It sets 1 to 7 day, uses preceding exhaustion PBS solution.Or it is trained by the coated cell of CD3 monoclonal antibody after freeze-drying removal PBS Feeding bottle can 4 DEG C of longer-term preservations.20ml training base is added before cell culture and cleans bottle wall, laying flat culture bottle makes to train base covering coating side Bottle wall stands 10 minutes, inhales and abandons training base, spare.
PD-1 closing CIK preparation: by 50ml heparin anti-coagulating inject 50ml centrifuge tube A in, at a temperature of 4 DEG C with 3000rpm is centrifuged 5min.Upper plasma is moved into 50ml centrifuge tube B, 56 DEG C of water-bath 30min, with 3000rpm at a temperature of 4 DEG C It is centrifuged 15min, collects supernatant, 4 DEG C save backup.
Hank ' s liquid or physiological saline are added into the centrifuge tube A to 48ml, mixes.Blood cell suspension is slowly added to To the Ficoll-Hypaque equipped with 4ml, (density: in 15ml centrifuge tube 1.077 ± 0.001), 8ml/ is managed, at a temperature of 4 DEG C 20min is centrifuged with 3000rpm.Collect mononuclearcell (PBMC) simultaneously move it into 1 50ml centrifuge tube, add Hank ' s liquid or Physiological saline is centrifuged 5min at a temperature of 4 DEG C with 3000rpm, abandons supernatant, be repeated 3 times to 50ml.
The above-mentioned sedimentation cell of 10ml anteserum-less substrate suspension is added, cell suspension is added to anti-human CD3 monoclonal antibody Coated 75cm2In culture bottle, in addition stating B pipe blood plasma 8ml, preferred blood plasma is patient's autologous plasma, can prevent allergic reaction, And anteserum-less substrate is added to 80ml, 37 DEG C, 5%CO2Under the conditions of cultivate 24 hours.IFN γ, IFN γ are added into culture bottle Final concentration of 1000U/ml, 37 DEG C, 5%CO2Under the conditions of cultivate 4-6 days or so.Microscopically observation cell covers with culture bottle When, cell is swept away from bottle wall using Pei Ji in measuring pipette and bottle and moves into 1500-2000ml cell come and by cell suspension In culture bag, addition 500ml anteserum-less substrate, the above-mentioned B pipe blood plasma of 10ml and IL-2 (final concentration of 300-500U/ml), 37 DEG C, 5%CO2Under the conditions of cultivate 4-5 days.Add anteserum-less substrate 1000ml and IL-2 (3 × 105-5×105U), 37 DEG C, 5%CO2Item It is cultivated 2 days under part.
It takes culture solution 20ml to carry out Bacteria Culture and detects mycoplasma using round pcr.Other cells are (thin in culture bag Born of the same parents) continue culture 2 days, cell is collected, 5min is centrifuged with 3000rpm at 4 DEG C, abandons supernatant.Physiological saline adds to 50ml, 4 5min is centrifuged with 3000rpm at DEG C, supernatant is abandoned, is repeated 3 times.
Add 20-40ml physiological saline, mix, the anti-human PD-1 monoclonal antibody of 200 μ g (final concentration of 50-250 μ g/ is added Ml), mix.(18 DEG C -25 DEG C) incubation 30min of room temperature.Adding concentration is the human serum albumins 4-10ml of 200mg/ml, adds physiology Salt water to 50ml, the filtering of 200 mesh filter screens moves into cell suspension in infusion bag, adds physiological saline to 300ml, heat sealing glues Labelling is used for venous re-transfusion (total number of cells: 1 × 108-1×109)。
Measure of merit
CIK and the closed CIK of PD-1 are taken, carries out the double dyeing of CD3, PD-1 immunofluorescence and cellulotoxic experiment respectively.CD3, The double Coloration experiments of PD-1 immunofluorescence: taking CIK and the closed each 0.5ml of CIK cell suspension of PD-1 to be added separately in 2 test tubes, 800rpm is centrifuged 5min, inhales and abandons supernatant, gently shakes, and mixes cell, it is mono- that the FITC label anti-human CD3 of mouse is added in each test tube simultaneously Clonal antibody and PE mark 100 μ l of rabbit-anti people PD-1 monoclonal antibody, mix, and are incubated at room temperature 30 minutes.5ml is added 0.01MPH7.4PBS is centrifuged 5min at 4 DEG C with 1000rpm, abandons supernatant, is repeated 3 times.It is outstanding that 1ml 0.01MPH7.4PBS is added Floating cell, flow cytomery, the PD-1 positive cell rate of CIK is 38.02% ± 16.43% before closing as the result is shown, envelope 3.12% ± 0.52% is down to after closing, PD-1 recall rate is substantially reduced (P < 0.05) after closing, and it is good to show that the method for the present invention has The effect of good closing PD-1 molecule, as a result as shown in Figure 1 and Figure 2.
Cellulotoxic experiment: with the full culture medium of RPMI1640 (calf serum, 100U/ml penicillin, 100U/ml containing 10% Streptomysin) the closed CIK of CIK, PD-1 is tuned into 1 × 105SCG7901 tumour cell is tuned into 1 × 10 by c/ml4c/ml.Respectively Above-mentioned cell is added in 96 porocyte culture plates.The hole A adds CIK cell or the closed CIK of PD-1,100 holes μ l/；The hole B adds SCG7901 tumour cell, 100 holes μ l/；After the every hole in the hole C adds the 100 closed CIK of μ l CIK or PD-1, it is swollen to add SCG7901 The every 100 μ l of hole of oncocyte.37 DEG C, 5%CO2Under the conditions of cultivate 72 hours, in each hole be added 5mg/ml MTT, 20 holes μ l/, continue Culture 4 hours.1000rpm is centrifuged 10min, inhales and abandons 180 μ l of supernatant, and DMSO is added, and 100 holes μ l/ mix, and microplate reader 450nm is surveyed Determine OD value.Killing rate calculation method is killing rate=[1- (C-A)/B] × 100%.Cyto toxic experiment showed shows CIK, PD-1 The tumor cell destruction of closed CIK is respectively as follows: 47.82% ± 12.24% and 73.3% ± 15.62%, and PD-1 is closed The tumor cell destruction of CIK is apparently higher than the tumor cell destruction (P < 0.05) of CIK, it was demonstrated that the tumour of PD-1 closing CIK Killing activity significantly improves, as a result as shown in Figure 3.

Claims (2)

1. the preparation method of the PD-1 closing CIK for treating tumour, characterized by comprising the steps of:
It is coated with step, anti-human CD3 monoclonal antibody coated cell culture bottle；
Step is activated, activates culture lymphocyte using the CD3 monoclonal antibody, IFN γ and IL-2；
Cleaning step, cleaning activate the lymphocyte after culture three times；
Step is closed, the lymphocyte after the cleaning that suspended with 20-50ml physiological saline obtains suspension cell solution, to institute Addition PD-1 monoclonal antibody in suspension cell solution is stated, 18-25 DEG C of suspension, which is incubated for 30 minutes, obtains cell suspension；
Include step: filtration step adds physiology salt after the filtering of 200 mesh filter screen of cell-dispersing agent is added in Xiang Suoshu cell suspension Water obtains feeding back liquid to 300ml；
The anti-human CD3 monoclonal antibody coated cell culture bottle comprises the following steps: using the PBS solution of 0.01M PH9.6 It dilutes first class and purifies anti-human CD3 monoclonal antibody to concentration 50-70ng/ml；It is mono- that the first class after dilution is purified into anti-human CD3 Clonal antibody is added in culture bottle, and 4 DEG C are placed 24 hours or more；
The activation step comprises the following steps:
Heparin anti-coagulating is injected in centrifuge tube A, 5min is centrifuged with 3000rpm at a temperature of 4 DEG C；Upper plasma is taken to move into centrifugation Pipe B, 56 DEG C of water-bath 30min are centrifuged 15min at a temperature of 4 DEG C with 3000rpm, collect supernatant, 4 DEG C save backup；Lower layer's blood Cell stays spare in centrifuge tube A；
Physiological saline suspends the haemocyte in the centrifuge tube A, and blood cell suspension obtained is slowly added into equipped with 4 In the centrifuge tube for the Ficoll-Hypaque that the density of ml is 1.077 ± 0.001, it is centrifuged at a temperature of 4 DEG C with 3000rpm 20min；It collects mononuclearcell confluent monolayer cells and the cell is cleaned three times using centrifugal process；Anteserum-less substrate is added to be resuspended, system Obtain anteserum-less substrate suspension cell；
The anteserum-less substrate suspension cell is added to containing in the anti-human coated culture bottle of CD3 monoclonal antibody, Inactivation blood plasma is added and adds into anteserum-less substrate, 37 DEG C, 5%CO2Under the conditions of cultivate 24 hours；IFN is added into culture bottle γ, 37 DEG C, 5%CO2Under the conditions of cultivate 4-6 days；Cell after taking culture moves into cell culture bags, and anteserum-less substrate is added, goes out Promoting blood circulation slurry and IL-2,37 DEG C, 5%CO2Under the conditions of cultivate 4-5 days；Add anteserum-less substrate and IL-2,37 DEG C, 5%CO2Under the conditions of Culture 4-5 days；
It takes culture solution 20ml to carry out Bacteria Culture and detects mycoplasma using round pcr；Cell in other cell, that is, culture bags Continue culture 2 days, collect cell, 5min is centrifuged with 3000rpm at 4 DEG C, abandons supernatant, physiological saline adds to 50ml, at 4 DEG C Under with 3000rpm be centrifuged 5min, abandon supernatant, be repeated 3 times；
Add 20-40ml physiological saline, mix, the anti-human PD-1 monoclonal antibody of 200 μ g: final concentration of 50-250 μ g/ml is added, mixes It is even；18 DEG C of -25 DEG C of incubation 30min of room temperature；Adding concentration is the human serum albumins 4-10ml of 200mg/ml, adds physiological saline extremely 50ml, 200 mesh filter screens filtering, cell suspension is moved into infusion bag, adds physiological saline to 300ml, heat sealing, label adhering Label are used for venous re-transfusion, total number of cells: 1 × 108-1×109。
2. the preparation method of the PD-1 closing CIK according to claim 1 for treating tumour, which is characterized in that in institute Also include before stating cleaning step: checking procedure detects cell quantity after carrying out cell culture, and utilizes round pcr detection culture Mycoplasma in supernatant.
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CN105861433A (en) * 2016-04-27 2016-08-17 天津普瑞赛尔生物科技有限公司 Method for preparing CIK cell preparation with high-efficiency tumor killing property and prepared CIK cell preparation

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