CN108392627A - The preparation method of DC-TAA tumor vaccines - Google Patents
The preparation method of DC-TAA tumor vaccines Download PDFInfo
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Abstract
The present invention provides a kind of preparation methods of DC TAA tumor vaccines, include the following steps：(1) peripheral blood mononuclear cells PBMC is isolated from the Cord blood of unbilical blood bank allotment type；(2) the fresh tumor tissues cut off after taking tumor patient to perform the operation obtain tumor cell lysis liquid, are prepared into tumor associated antigen TAA；(3) CIK is induced into jointly with cytokine profiles in vitro, CIK cell is obtained after 12~15d induced amplifications, then after strict quality control detection is qualified, point 6 feedback patient's bodies；(4) in the laboratories GMP, the 5th day of culture is trained PBMC at antigen presenting cell DC for a certain area with cytokine profiles, and after antigen presenting cell DC is ripe by the identification of cell table, the tumour antigen TAA of preparation and its carrier cell DC are incubated altogether；It is prepared into DC TAA tumor vaccines after irradiation inactivation in (5) the 8th days；(6) peripheral blood venous re-transfusion is carried out under the operation of clinician or is injected to the Local Multipoint of patient part superficial lymph tie region.
Description
Technical field
The present invention relates to a kind of preparation methods of biological products, belong to biotechnology adoptive immunity application category, i.e.,
For the preparation method of tumour antigen vaccine.
Background technology
Cellular immunotherapy is a kind of emerging, with significant curative effect tumor treatment model, is that a kind of autoimmunity is anti-
The novel method for the treatment of of cancer.It is carried out in vitro to the immunocyte acquired from the patient with biotechnology and biological agent
Method in patient body is fed back to after culture and amplification, to excite, enhances body autoimmune function, to reach treatment tumour
Purpose.Biological immune treatment is the fourth-largest oncotherapy technology after operation, radiation and chemotherapy.There is killing in body
The lymphocyte of effect has natural killer cells, the cell killing T cell of killing etc., themselves can be to antitumor cell
Generation.According to Germicidal efficacy, a tumour cell needs a lymphocytes up to a hundred to tackle it.And the tumor of one cubic centimetre of size
There are about 1,000,000,000 oncocytes in block.Therefore, if there is a large amount of lymphocyte, it will be able to effective tumors destroyed cell, confrontation
The generation of tumour cell, here it is the basic concepts of cellular immunotherapy.
Currently, the cell biological headed by specificity antineoplastic immunity therapy treats oneself through displaying talent for the first time, and becomes tumour life
Important developing direction in object treatment.Cellular immunotherapy, full name are adoptive immunity cell therapy (adoptive
Cellular immunotherapy, ACI or AIT), it is directed to tumor patient and transmits immunocyte with anti-tumor activity
(specific and nonspecific), the immune response killing tumor cell of direct killing tumour or excitating organism.Refer to clinically
The self or alloimmune effector cell of Activation In Vitro is transfused to patient, is controlled with killing one kind of tumour cell of patient's body
Treatment mode.It, can application cell immunization therapy prevention recurrence and transfer after primary tumor is dispelled (operation, radiotherapy)；Tumour shifts extensively,
Operator can not be carried out, can be used with Chemotherapy plus；In the chemotherapy intermittent phase, application cell immunization therapy can restore machine as early as possible
The impaired immune function of body can also increase chemotherapy and the effect of immunization therapy simultaneously with the application of certain chemotherapy drugs in combination.It is certain
Immunocyte such as CIK cell and DC cells etc. still have lethal effect to the tumour cell for generating drug resistant gene.It is unwise to chemicotherapy
Sense person or the tumor patient that can not be resistant to；Part is not suitable for performing an operation, the patients with advanced cancer of interventional treatment and other treatment into
Row cellular immunotherapy can improve the immune function of patient, improve quality of life, elongated strap tumor life cycle；Some patients pass through
The comprehensive cellular immunotherapy of large dosage can obviously reduce the volume of tumour, strive for operation or other treatment chance, a small number of late periods
Tumour patient is by can also reach partly or completely direct release after cellular immunotherapy.Cellular immunotherapy optionally inhibits/
Killing tumor cell, and independent of the immune function of lotus knurl person, and being shared with Radiotherapy chemotherapy, from the eighties in last century with
To have been carried out numerous studies.The therapy after NK, gamma delta T, CD3AK, DC-CIK, LAK, TIL, CIK, the development rank such as EAAL
The case where section, curative effect, specificity, whole efficient, side effect reaction etc., gradually reduces.Tumour for loading DC is anti-
Original can be tumour specific antigen peptide (Tumor-Specific Antigens, TSA) or tumor associated antigen (Tumor-
Associated Antigens, TAA), can also be the full cellular antigens of tumour.Have with the DC that TSA or TAA is loaded good
Targeting, but there is this method fixed tumour specific antigen or the few immune attack with single antigen of Antigenic Peptide type to pass through
The defects of Chang Wufa killing tumor cells.And can overcome these defects with the full cellular antigens load DC of tumour, because being not necessarily at this time
Know which antigen is the TSA or TAA of tumour cell, and a variety of different tumour antigen impact DC in holoantigen can induce production
The raw Cytotoxic T lymphocytes (CTL) for different antigenic determinants are cloned, to realize effective killing to tumour cell.
" tumor seedling " (tumor vaccine), the tumor vaccine of the usual prior art are first to be prepared to control with malignant tumour by scientific research personnel
Cytokine gene cDNA needed for treating, is cloned into safety retroviral vector, and be packaged into the deficiency of recombination
Retroviral particle forms " tumor seedling " to infected tumor's cell, and there are the preparation methods of many tumor seedlings for the prior art.Patent
Method is disclosed in numbers 97105061.9：Take autologous tumor tissue specimens paraffin embedding slices, egg cell suspension be made, through allicin,
Add BCG vaccine adjuvant and aldehyde fixative after extract solution from aloe processing, however this autovaccine material source is limited, and group
The curative effect after applying can be influenced by knitting the difference of material source, and curative effect also has apparent difference caused by the position of different dosing,
Such as：Venous re-transfusion and regional nodes' injection are just different, and the observation of clinical efficacy duration, the patient after has height
Heat symptom-complex shape, while being also not suitable for the application of part lymphsystem tumor patient.
Invention content
In order to overcome the missing and deficiency of this part in the prior art, the present invention to provide a kind of system of DC-TAA tumor vaccines
Preparation Method includes the following steps：
(1) peripheral blood mononuclear cells PBMC is isolated from the Cord blood of unbilical blood bank allotment type；
(2) the fresh tumor tissues cut off after taking tumor patient to perform the operation obtain tumor cell lysis liquid, are prepared into tumour
Related antigen TAA；
(3) CIK is induced into jointly with cytokine profiles in vitro, it is thin that CIK is obtained after 12~15d induced amplifications
Born of the same parents, then after strict quality control detection is qualified, point 6 feedback patient's bodies；
(4) in the laboratories GMP, being trained for a certain area PBMC with cytokine profiles for the 5th day for culture is thin at antigen submission
Born of the same parents DC, after antigen presenting cell DC is ripe by the identification of cell table, by the tumour antigen TAA of preparation and its carrier cell DC
It is incubated altogether；
It is prepared into DC-TAA tumor vaccines after irradiation inactivation in (5) the 8th days；
(6) peripheral blood venous re-transfusion or the office to patient part superficial lymph tie region are carried out under the operation of clinician
Portion's multi-point injection.
Preferably, it includes step that the step (1), which isolates peripheral blood mononuclear cells PBMC,：
(1-1) acquires the peripheral blood mononuclear cells 50-100mL of patient itself with blood cell separator；
Peripheral blood mononuclear cells PBMC is further purified using lymphocyte separation medium density-gradient centrifugation method in (1-2)；
(1-3) serum-free medium washs 2 times, obtains the high-purity peripheral blood mononuclear cells of certain cell quantity
PBMC, the cell quantity need to reach certain standard.
Preferably, for the purity of the step (1-3) 90% or more, the cell quantity reaches 1-3 × 108。
Preferably, the step (2) the acquisition tumor cell lysis liquid specifically includes：
(2-1) ocal resection sample under aseptic condition, nonneoplastic tissue by slough and cancer is removed clean；
(2-2) sterile saline is washed 3 times；
(2-3) is shredded tumor tissues with sterile tissue shear, and RPMI1640 culture mediums are added, are fully ground；
Single cell suspension is collected after the sterile net filtration of (2-4) 200 mesh；
Cell is resuspended to 1-2 × 10 with RPMI1640 culture mediums in (2-5)7/ ml is fitted into the sterile cryopreservation tubes of 5ml；
(2-6) will be quick-frozen in cryopreservation tube immersion liquid nitrogen, takes out after ten minutes, then be put into rapidly in 37 DEG C of water-baths and thaw 10 points
Clock, 3-5 times repeatedly；
(2-7) Tumor lysate is added in centrifuge tube, is centrifuged 10 minutes with 3000rpm rotating speeds；
(2-8) collects supernatant, 0.22mm membrane filtrations degerming or after the inactivation of cobalt -60, and keep sample detection protein content and thin
Bacterium, fungi and mycoplasma；
- 80 DEG C of (2-9) is saved backup.
Preferably, the step (2-6) can also use following operation：Cryopreservation tube is put into it is quick-frozen in -80 DEG C of environment, 10
It is taken out after minute, then is put into rapidly in 37 DEG C of water-baths and thaws 10 minutes, repeatedly 3-5 times.
Preferably, the step (3) includes：
PBMC is pressed 1-2 × 10 by (3-1)6The concentration of/ml is suspended in serum-free medium, and the weight of 1000U/ml is added
Group people's IFN-γ, 37 DEG C, 5%CO2It is cultivated in incubator；
The recombinant human il-2 of the CD3 monoclonal antibodies and 300U/ml of 50ng/ml is added in (3-2) after 24 hours, stimulate CIK
The growth of cell and proliferation；
(3-3) every 3 days half amounts change liquid or expansion bottle is primary, and add recombinant human il-2 300U/ml；
(3-4) harvests CIK cell in the 14d of culture.
Preferably, the step (3) further includes step (3-5) CIK cell Quality Control, wherein specifically including：
(3-5-1) Trypan Blue detects：Wherein living cells should be 80% or more；
The equimolecular expression of (3-5-2) flow cytomery cell surface CD3, CD8, CD56：Wherein D3+CD56+ is thin
The ratio of born of the same parents should be 20% or more；
(3-5-3) cell killing is tested：It is effector cell with CIK cell, using tumour cell as target cell, by effector cell
10 are pressed with target cell:1 number than ratio be added 96 hole U-shaped boards in, per hole contain target cell 1 × 104A, final volume is 200 μ
L cultivates 4h, then takes culture supernatant if 3 multiple holes, and effector cell is detected to target cell with lactate dehydrogenase L DH kits
Killing rate；
Before (3-5-4) harvests cell, a small amount of culture is taken to carry out bacterium, fungal culture, and detect mycoplasma, Chlamydia and
Endotoxin.
Preferably, the recombined human IL-1 α of 100U/ml can be added in the step (3-2) simultaneously.
Preferably, the tumour cell of the step (3-5-3) is primary tumor cell or tumor cell line.
Preferably, the standard of the detection of the step (3-5-4) is：Pathogeny detection is negative, endotoxin<5Eu.
Preferably, it is (5~8) × 10 to feed back total number of cells in the step (3) per the course for the treatment of9It is a.
Preferably, step (5) inactivation uses cobalt -60, and 24-48 hours after the completion of step (4).
Specific implementation mode
Tumour antigen for loading DC can be tumour specific antigen peptide (Tumor-Specific
Antigens, TSA) or tumor associated antigen (Tumor-Associated Antigens, TAA), can also be swollen
The full cellular antigens of tumor.There is good targeting with the DC that TSA or TAA is loaded, but this method has fixed tomour specific
Property antigen or Antigenic Peptide type is few and the immune attack of single antigen often can not killing tumor cell the defects of.And it is complete with tumour
Cellular antigens load DC can overcome these defects, because being not necessarily to know which antigen is the TSA or TAA of tumour cell at this time, and
And the inducible generations of a variety of different tumour antigen impact DC in holoantigen are thin for the cytotoxic T lymph of different antigenic determinants
Born of the same parents (CTL) clone, to realize effective killing to tumour cell.There are many method that tumour cell holoantigen loads DC, including
DC is loaded with tumor cell lysis liquid load DC, with apoptotic tumor cell load DC, with necrosis or dead tumour cell, with swollen
Tumor living cells loads DC, and tumour cell is merged with DC.Clinically the most commonly used is loaded with tumor cell lysis liquid at present
DC, because this method is simple, quick, effective.Multigelation is the common methods for obtaining tumor cell lysis liquid.
The preparation method of DC-TAA tumor vaccines, includes the following steps：
(1) peripheral blood mononuclear cells PBMC, including step are isolated from the Cord blood of unbilical blood bank allotment type：
(1-1) acquires the peripheral blood mononuclear cells 50-100mL of patient itself with blood cell separator；
Peripheral blood mononuclear cells PBMC is further purified using lymphocyte separation medium density-gradient centrifugation method in (1-2)；
(1-3) serum-free medium washs 2 times, obtains the high-purity peripheral blood mononuclear cells of certain cell quantity
PBMC, the cell quantity need to reach certain standard, i.e., for purity 90% or more, the cell quantity reaches 1-3 × 108；
(2) the fresh tumor tissues cut off after taking tumor patient to perform the operation obtain tumor cell lysis liquid, are prepared into tumour
Related antigen TAA obtains tumor cell lysis liquid and specifically includes：
(2-1) ocal resection sample under aseptic condition, nonneoplastic tissue by slough and cancer is removed clean；
(2-2) sterile saline is washed 3 times；
(2-3) is shredded tumor tissues with sterile tissue shear, and RPMI1640 culture mediums are added, are fully ground；
Single cell suspension is collected after the sterile net filtration of (2-4) 200 mesh；
Cell is resuspended to 1-2 × 10 with RPMI1640 culture mediums in (2-5)7/ ml is fitted into the sterile cryopreservation tubes of 5ml；
(2-6) will be quick-frozen in cryopreservation tube immersion liquid nitrogen, takes out after ten minutes, then be put into rapidly in 37 DEG C of water-baths and thaw 10 points
Clock 3-5 times repeatedly, can also use following operation：Cryopreservation tube is put into quick-frozen in -80 DEG C of environment, taken out after ten minutes, then fast
Speed, which is put into 37 DEG C of water-baths, thaws 10 minutes, repeatedly 3-5 times；
(2-7) Tumor lysate is added in centrifuge tube, is centrifuged 10 minutes with 3000rpm rotating speeds；
(2-8) collects supernatant, 0.22mm membrane filtrations degerming or after the inactivation of cobalt -60, and keep sample detection protein content and thin
Bacterium, fungi and mycoplasma；
- 80 DEG C of (2-9) is saved backup；
(3) CIK is induced into jointly with cytokine profiles in vitro, it is thin that CIK is obtained after 12~15d induced amplifications
Born of the same parents, then after strict quality control detection is qualified, point 6 feedback patient's bodies, it is (5~8) × 10 that total number of cells are fed back per the course for the treatment of9
It is a, including step：
PBMC is pressed 1~2 × 10 by (3-1)6The concentration of/ml is suspended in serum-free medium, and the weight of 1000U/ml is added
Group people's IFN-γ, 37 DEG C, 5%CO2It is cultivated in incubator；
The recombinant human il-2 of the CD3 monoclonal antibodies and 300U/ml of 50ng/ml is added in (3-2) after 24 hours, stimulate CIK
The recombined human IL-1 α of 100U/ml can be also added in the growth of cell and proliferation simultaneously at this time；
(3-3) every 3 days half amounts change liquid or expansion bottle is primary, and add recombinant human il-2 300U/ml；
(3-4) harvests CIK cell in the 14d of culture；
(3-5) CIK cell Quality Control, wherein specifically including：
(3-5-1) Trypan Blue detects：Wherein living cells should be 80% or more；
The equimolecular expression of (3-5-2) flow cytomery cell surface CD3, CD8, CD56：Wherein D3+CD56+ is thin
The ratio of born of the same parents should be 20% or more；
(3-5-3) cell killing is tested：It is effector cell with CIK cell, using tumour cell as target cell, tumour cell is
Effector cell and target cell are pressed 10 by primary tumor cell or tumor cell line:1 number than ratio be added 96 hole U-shaped boards
In, target cell 1 × 10 is contained per hole4A, final volume is 200 μ l, if 3 multiple holes, cultivates 4h, then takes culture supernatant, use lactic acid
Dehydrogenase LDH kits detect killing rate of the effector cell to target cell；
Before (3-5-4) harvests cell, a small amount of culture is taken to carry out bacterium, fungal culture, and detect mycoplasma, Chlamydia and
The standard of endotoxin, detection is：Pathogeny detection is negative, endotoxin<5Eu.
(4) in the laboratories GMP, being trained for a certain area PBMC with cytokine profiles for the 5th day for culture is thin at antigen submission
Born of the same parents DC, after antigen presenting cell DC is ripe by the identification of cell table, by the tumour antigen TAA of preparation and its carrier cell DC
It is incubated altogether；
DC-TAA tumor vaccines are prepared into after irradiation inactivation in (5) the 8th days, inactivation uses cobalt -60, and complete in step (4)
At 24-48 hours latter；
(6) peripheral blood venous re-transfusion or the office to patient part superficial lymph tie region are carried out under the operation of clinician
Portion's multi-point injection.
The advantage of the tumor seedling is：1) administration route is converted：There is high fever using Most patients after above-mentioned technical method
Phenomenon, such as some critical patients cannot be subjected to the process of fever, so our changing by cell culture technology
It is good, the purity of carrier cell is further purified, after being then incubated altogether with antigen, administration route is become by peripheral blood venous re-transfusion
It is injected for the Local Multipoint of local superficial lymph tie region, thus reduces the adverse reaction of patient, while also improving immune
The effect for the treatment of；2) cell quantity is precisely controlled：Adoptive immunotherapy principle is exactly to improve the immune defense of patient, and foundation is exempted from
Epidemic disease defense system, then it is a kind of normal phenomenon that the fever of patient, which also belongs to, only some patient's bodies do not tolerate.So sick
The cell concentration that the temperature of human hair heat feeds back or injects with cell is related.Each cell quantity 3.0~5.0 × 108For safety value, such as
1 time infusion amount is more than the numerical value, then sick person's development can be apparent.So to be repeatedly principle on a small quantity.
In the present embodiment, the dendritic cells (DcTAA) using observation autologous tumor antigens load combine distribution type derived from cord blood
Cytokine induced kill cell (CIK) clinical efficacies of combination therapy 48 medium and advanced lung cancers is immunized.Method, which uses, matches
The bleeding of the umbilicus separation mononuclearcell (PBMC) of type uses cytokine profiles [CD3McAb, interleukins (IL) -2, to swell in vitro
Induction is obtained at CIK and dendritic cells (DC) after 12~15d induced amplifications jointly by tumor necrosis factor (IFN)-γ, IL-1d etc.
CIK cell, then after strict quality control detection is qualified, point 6 feedback patient's bodies, fed back per the course for the treatment of total number of cells be (5~
8)×109It is a.The autologous tumor antigens of the 5th day of culture load DC, and the 8th day harvest DCTAA, row lymph node position is subcutaneously noted
It penetrates, the size of knurl, clinical symptoms integral, quality of life and amynologic index, Kamofsky are commented after observation treatment patients
Point, weight, adverse reaction etc. variation, while recording the life cycle of patient.As a result 48 receive bleeding of the umbilicus DCTAA-CIK treatments
Patient in, it is 37 that complete incidence graph (CR)+part, which alleviates (PR), Overall response rate 77.1%.Symptom scores improvement rate
78.9%~84.7%；Kamofsky scores increase rate as 89.6% (43/48).1 year survival rate 80.6%.Adverse reaction is light
It is micro-.DCTAA-CIK cell therapy peripheral blood in patients CD3, cd4 t cell and NK cell proportions be significantly increased (42.21 ±
6.12) %, (24.42 ± 3.01) %, 0.99 ± 0.34, (24.98 ± 3.02) % and (71.58 ± 7.64) %, (37.25 ±
2.13) %, 1.62 ± 0.45, (35.23 ± 4.11) %, t values are respectively 6.34,5.67,0.25 and 4.43, and P values are equal<0.01.
So as to judge that DCTM and the CIK cell adoptive immunotherapy of the present invention of derived from cord blood are treatment middle and advanced stages
A kind of good method of lung cancer can significantly improve patient immune function, improve patient clinical symptom, improve life quality, prolong
Longevity period.
It, will not be by these embodiments although the present invention is described by reference to specific illustrative embodiment
Restriction and only limited by accessory claim.It should be understood by those skilled in the art that can be without departing from the present invention's
The embodiment of the present invention can be modified and be changed in the case of protection domain and spirit.
Claims (12)
1. a kind of preparation method of DC-TAA tumor vaccines, it is characterised in that include the following steps：
(1) peripheral blood mononuclear cells PBMC is isolated from the Cord blood of unbilical blood bank allotment type；
(2) the fresh tumor tissues cut off after taking tumor patient to perform the operation obtain tumor cell lysis liquid, are prepared into tumour correlation
Antigen TAA；
(3) CIK is induced into jointly with cytokine profiles in vitro, CIK cell is obtained after 12~15d induced amplifications, then
After strict quality control detection is qualified, point 6 feedback patient's bodies；
(4) in the laboratories GMP, the 5th day of culture is trained PBMC at antigen presenting cell DC for a certain area with cytokine profiles,
After antigen presenting cell DC is ripe by the identification of cell table, the tumour antigen TAA of preparation and its carrier cell DC are incubated altogether
It educates；
It is prepared into DC-TAA tumor vaccines after irradiation inactivation in (5) the 8th days；
(6) peripheral blood venous re-transfusion or more to the part of patient part superficial lymph tie region is carried out under the operation of clinician
Point injection.
2. a kind of preparation method of DC-TAA tumor vaccines according to claim 1, it is characterised in that the step (1) point
It includes step to separate out peripheral blood mononuclear cells PBMC：
(1-1) acquires the peripheral blood mononuclear cells 50-100mL of patient itself with blood cell separator；
Peripheral blood mononuclear cells PBMC is further purified using lymphocyte separation medium density-gradient centrifugation method in (1-2)；
(1-3) serum-free medium washs 2 times, obtains the high-purity peripheral blood mononuclear cells PBMC of certain cell quantity, institute
Certain standard need to be reached by stating cell quantity.
3. a kind of preparation method of DC-TAA tumor vaccines according to claim 2, it is characterised in that the step (1-3)
Purity 90% or more, the cell quantity reaches 1-3 × 108.
4. a kind of preparation method of DC-TAA tumor vaccines according to claim 1, it is characterised in that step (2) institute
Acquisition tumor cell lysis liquid is stated to specifically include：
(2-1) ocal resection sample under aseptic condition, nonneoplastic tissue by slough and cancer is removed clean；
(2-2) sterile saline is washed 3 times；
(2-3) is shredded tumor tissues with sterile tissue shear, and RPMI1640 culture mediums are added, are fully ground；
Single cell suspension is collected after the sterile net filtration of (2-4) 200 mesh；
Cell is resuspended to 1-2 × 107/ml with RPMI1640 culture mediums in (2-5), is fitted into the sterile cryopreservation tubes of 5ml；
(2-6) will be quick-frozen in cryopreservation tube immersion liquid nitrogen, takes out after ten minutes, then be put into rapidly in 37 DEG C of water-baths and thaw 10 minutes,
3-5 times repeatedly；
(2-7) Tumor lysate is added in centrifuge tube, is centrifuged 10 minutes with 3000rpm rotating speeds；
(2-8) collects supernatant, 0.22mm membrane filtrations degerming or after the inactivation of cobalt -60, keep sample detection protein content and bacterium, true
Bacterium and mycoplasma；
- 80 DEG C of (2-9) is saved backup.
5. a kind of preparation method of DC-TAA tumor vaccines according to claim 4, it is characterised in that the step (2-6)
Following operation can also be used：Cryopreservation tube is put into quick-frozen in -80 DEG C of environment, taken out after ten minutes, then be put into 37 DEG C of water rapidly
It thaws 10 minutes, repeatedly 3-5 times in bath.
6. a kind of preparation method of DC-TAA tumor vaccines according to claim 1, it is characterised in that step (3) packet
It includes：
PBMC is suspended in by the concentration of 1~2 × 106/ml in serum-free medium by (3-1), and the recombined human of 1000U/ml is added
IFN-γ, is cultivated in 5%CO2 incubators by 37 DEG C；
The recombinant human il-2 of the CD3 monoclonal antibodies and 300U/ml of 50ng/ml is added in (3-2) after 24 hours, stimulate CIK cell
Growth and proliferation；
(3-3) every 3 days half amounts change liquid or expansion bottle is primary, and add recombinant human il-2 300U/ml；
(3-4) harvests CIK cell in the 14d of culture.
7. a kind of preparation method of DC-TAA tumor vaccines according to claim 6, it is characterised in that the step (3) is also
Including step (3-5) CIK cell Quality Control, wherein specifically including：
(3-5-1) Trypan Blue detects：Wherein living cells should be 80% or more；
The equimolecular expression of (3-5-2) flow cytomery cell surface CD3, CD8, CD56：Wherein D3+CD56+ cells
Ratio should be 20% or more；
(3-5-3) cell killing is tested：It is effector cell with CIK cell, using tumour cell as target cell, by effector cell and target
Cell presses 10:1 number than ratio be added 96 hole U-shaped boards in, per hole contain target cell 1 × 104, final volume be 200 μ l, if
3 multiple holes cultivate 4h, then take culture supernatant, the killing with lactate dehydrogenase L DH kits detection effector cell to target cell
Rate；
Before (3-5-4) harvests cell, a small amount of culture is taken to carry out bacterium, fungal culture, and detect mycoplasma, Chlamydia and endogenous toxic material
Element.
8. a kind of preparation method of DC-TAA tumor vaccines according to claim 6, it is characterised in that the step (3-2)
The recombined human IL-1 α of 100U/ml can be added simultaneously.
9. a kind of preparation method of DC-TAA tumor vaccines according to claim 7, it is characterised in that the step (3-5-
3) the tumour cell is primary tumor cell or tumor cell line.
10. a kind of preparation method of DC-TAA tumor vaccines according to claim 7, it is characterised in that the step (3-
The standard of detection 5-4) is：Pathogeny detection is negative, endotoxin<5Eu.
11. a kind of preparation method of DC-TAA tumor vaccines according to claim 1, it is characterised in that the step (3)
In per the course for the treatment of feed back total number of cells be (5~8) × 109.
12. a kind of preparation method of DC-TAA tumor vaccines according to claim 1, it is characterised in that the step (5)
Inactivation uses cobalt -60, and 24-48 hours after the completion of step (4).
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