CN103937741A - Preparation method of enhanced CIK (Cytokine Induced Killer) cell, and cell preparation - Google Patents
Preparation method of enhanced CIK (Cytokine Induced Killer) cell, and cell preparation Download PDFInfo
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Abstract
The invention discloses a preparation method of an enhanced CIK cell, and a cell preparation. The method comprises the following steps: separating to obtain a peripheral blood mononuclear cell from peripheral blood collected from a sufferer; putting the peripheral blood mononuclear cells into a blood-free medium to culture, so as to obtain karyocyte of which the concentration is (1-3)*10<6>/ml; adding IFN (interferon)-gamma, phytohemagglutinin and PGE2 (Phenyl Glycidyl Ether), transferring to a culture bottle or a culture bag to culture for 24 hours, and then adding a CD3 monoclonal antibody, IL-2 (interleukin-2), IL-1alpha, IL-4 and GM-CSF (granulocyte-macrophage colony-stimulating factor); continuing to culture for 3-5 hours, and then controlling the cell density at (1-6)*10<6>/ml; and centrifugally colleting to obtain the enhanced CIK cell after continuously culturing for the 14th to 21st days. The enhanced CIK cell preparation comprises the enhanced CIK cell, human serum albumin, IL-2, amino acids, vitamins and inorganic salts. Compared with a preparation in the patient, the CIK preparation has the advantages that the preservation time of the CIK preparation is obviously prolonged, and is prolonged to 36 hours from 6 hours at room temperature. The CIK preparation is higher in reliability in clinical application, and the safety is effectively ensured.
Description
Technical field
The present invention relates to immunocyte in vitro culture field, in particular to a kind of enhanced CIK cell preparation method and cell preparation.
Background technology
Adoptive cellular immunotherapy refers to the treatment method by being transfused itself or homospecificity, non-specific antineoplastic immune effector cell, direct killing tumour cell or virus.Oneself enters clinical practice to adoptive cellular immunotherapy, and as after the fourth-largest tumor therapeuticing method after operation, radiotherapy, chemotherapy.The species of such immunocyte includes Tumor-infiltrating lymphocytes (LAK), tumor-infiltrating lymphocytes (TIL), cellulotoxic lymphocyte (CTL) etc., in numerous immunocytes, CIK cell is high due to killing tumor activity, kill knurl spectrum wide, it is same to a variety of mdr cells sensitive and to the good characteristics such as normal marrow hemopoiesis precursor cells toxicity is smaller, it is widely used in clinic.
Compared with other immunocytes, CIK cell be oneself know to one of tumor cytotoxicity activity most strong effector cell.It is the synergy by cytokine profiles, by PMNC culture Lai a group foreign cell.The quantity and cytotoxic activity of CIK cell directly determine its effect in clinical treatment.NKT cells are the most cells of cytotoxic activity in CIK foreign cells group, and its cell surface expresses two kinds of membrane protein molecules of CD3 and CD56 simultaneously（That is CD3+And CD56+）, both with the powerful antitumor activity of T lymphocytes but also with NK cells non-MHC it is restricted kill knurl the characteristics of.However, NKT cell contents are extremely low in the peripheral blood of people, only 1% or so.According to Germicidal efficacy, removing a tumour cell needs up to a hundred lymphocytes.And 1cm31,000,000,000 oncocytes are there are about in the tumor mass of size.Therefore, the effect for killing tumour is unable to reach by the immunocyte of itself, the absolute quantity of this kind of cell by vitro culture, can only be improved.
CIK cell can largely be bred by external evoked, general CIK preparation methods are after the PMNC (PBMC) of separation is handled 24 hours with IFN-γ, add CD3mAb, IL-1 α and the IL-2 factor carry out stimulation induction, a number of CIK cell is finally obtained, but the increment multiple and cytotoxic activity of the CIK cell finally obtained are all not ideal enough.
The content of the invention
The technical problems to be solved by the invention are just to provide a kind of high proliferation power, the enhanced CIK cell formulation preparation method and cell preparation of high cytotoxic activity, the CIK preparations when stored between on be significantly increased.Brought up to 36 hours from 6 hours at room temperature.Reliability is higher in clinical practice, and security obtains effective guarantee.
In order to solve the above technical problems, a kind of preparation method for enhanced CIK cell that the present invention is provided, comprises the following steps：
1）The isolated PMNC from the peripheral blood of collection patient；It is placed in blood-free medium and cultivates, obtains 1~3 × 106Individual/ml mononuclearcell, adds IFN-γ, phytohemagglutin phytolectin and PGE2 and is transferred in blake bottle or culture bag, is 37 DEG C, 5%CO in temperature2, cultivate 24h under the conditions of saturated humidity；
Wherein：The concentration of the IFN-γ：100~2000u/ml, PHA concentration：10ng~50 μ g/ml, PGE2 concentration：The μ g/ml of 1ng~50；
2）Then CD3 monoclonal antibodies are added, IL-2, IL-1 α, IL-4 and GM-CSF continue to cultivate；The concentration of CD3 monoclonal antibodies：1ng~20 μ g/ml, IL-2 concentration：10~10000IU/ml, IL-1 α concentration：The μ g/ml of 1ng~50；IL-4 concentration：The μ g/ml of 1ng~1；GM-CSF concentration is 100~10000U/ml；
3）After culture 3~5 days, addition contains IL-2 serum free mediums, and control cell density is 1~6 × 106/ ml, wherein：The concentration of the IL-2：10~10000IU/ml；
4）Beginning in 7th day was counted every 2~3 days cells to culture, and added the serum free medium containing IL-2 and sodium selenite according to cell concentration, after continuously cultivating the 14th~21 day, is collected by centrifugation and is obtained enhanced CIK cell, wherein：The concentration of the IL-2 is 10~10000IU/ml, and the concentration of sodium selenite is 1 μ g~1mg/L.
Further, the cell density of the enhanced CIK cell：1~6 × 106-8Individual/ml
Preferably, the preparation method of enhanced CIK cell, comprises the following steps：
1）The isolated PMNC from the peripheral blood of collection patient；It is placed in blood-free medium and cultivates, obtains 1 × 106Individual/ml nucleus, adds IFN-γ, phytohemagglutin phytolectin and PGE2 and is transferred in blake bottle or culture bag, is 37 DEG C, 5%CO in temperature2, cultivate 24h under the conditions of saturated humidity；Wherein：The concentration of the IFN-γ：2000IU/ml, PHA concentration：100ng/ml, PGE2 concentration：50μg/ml；
2）Then CD3 monoclonal antibodies are added, IL-2, IL-1 α, IL-4 and GM-CSF continue to cultivate；The concentration of CD3 monoclonal antibodies：20 μ g/ml, IL-2 concentration：1000IU/ml, IL-1 α concentration：50ng/ml；IL-4 concentration：100ng/ml；GM-CSF concentration is 2000U/ml；
3）After culture 3~5, addition contains IL-2 serum free mediums, and control cell density is 1 × 106/ ml, wherein：The concentration of the IL-2：1000IU/ml；
4）Beginning in 7th day was counted every 2~3 days cells to culture, and added the serum free medium containing IL-2 and sodium selenite according to cell concentration, after continuously cultivating the 14th~21 day, and it is 1~6 × 10 to be collected by centrifugation and obtain cell density6Individual/ml enhanced the CIK cell of enhanced CIK cell, wherein：The concentration of the IL-2 is 1000IU/ml, and the concentration of sodium selenite is 100 μ g/L.
Present invention also offers a kind of enhanced CIK cell preparation, enhanced CIK cell, human serum albumin, IL-2, amino acid, vitamin and the inorganic salts obtained using the preparation method of the enhanced CIK cell described in any one in claims 1 to 3.
Further, the human serum albumin concentration：1~300g/L, IL-2 concentration：L~9 × 105~7U/ml, amino acid concentration：50mg~10g/L, vitamine concentration：100ug~50mg/L, inorganic salt concentration：500mg~10g/L.
Yet further, the human serum albumin concentration：50~200g/L, IL-2 concentration：L~9 × 105~6U/ml, amino acid concentration：500mg~5g/L, vitamine concentration：1~30mg/L, inorganic salt concentration：1g~7g/L.
Yet further, the human serum albumin concentration：100g/L, IL-2 concentration：2×106U/ml, amino acid concentration：3g/L, vitamine concentration：15mg/L, inorganic salt concentration：4g/L.
Yet further, the amino acid is any five kinds or more than five kinds in L-arginine, L-ASPARTIC ACID, Pidolidone, L-Histidine, ILE, L lysine HCL, L-phenylalanine, Serine, L-Trp, Valine, L- asparagines, CYSTINE dihydrochloride, glycine, L- hydroxyprolines, L-Leu, METHIONINE, L-PROLINE, L-threonine and TYR；
The vitamin is any three kinds or more in folic acid, riboflavin, biotin, p-aminobenzoic acid, puridoxine hydrochloride, vitamin B12 and niacinamide；
The inorganic salts be in sodium chloride, potassium chloride, AMSP, calcium nitrate and sodium acid carbonate it is any two or more.
The beneficial effects of the present invention are：
1st, CIK cell can largely be bred by external evoked, general CIK preparation methods are after the PMNC (PBMC) of separation is handled 24 hours with IFN-γ, add CD3mAb, IL-1 α and the IL-2 factor carry out stimulation induction, a number of CIK cell is finally obtained, but the increment multiple and cytotoxic activity of the CIK cell finally obtained are all not ideal enough.
2nd, it was found that adding IFN-γ stimulates in CIK cell incubation, PBMC can occur apoptosis, have impact on the amplification of cell.And PHA is essentially identical to PBMC inducing effect and IFN-γ, and with efficient proliferation, can as T cell strong activator, promote immunocompetent cell precursor to be converted to immunocompetent cell.Therefore substitute part IFN-γ to improve the increment multiple of CIK cell with PHA in the present invention.
3rd, CD4+CD25+Treg cells suppress the immunologic function of the T cell of activation by the CTLA-4 of activating T cell.And PGE2 can suppress mononuclearcell to CD4+CD25+Treg cell differentiations.Therefore the effect of activation CIK cell is strengthened with PGE2 in the present invention.
4th, IL-4 and GM-CSF have synergy, and phagocytosis, the killing activity of Neutrophil-mediated can be strengthened while PBMC multiplication capacities are strengthened.
5th, sodium selenite can improve the toxicity of CIK cell, therefore in the present invention, we add sodium selenite in CIK cell culture, to improve lethal effect of the final CIK cell to tumour cell.
6th, the CIK cell preparation clinically applied is typically CIK cell, human serum albumin, IL-2 and NaCl.Whole system is not appropriate for CIK cell and maintained vigour, and preparation must be fed back in patient's body after preparing in 6 hours, and otherwise CIK cell vigor is significantly reduced, and loses therapeutic action.After this method is used containing several amino acids, the nutrient solution of inorganic salts, CIK cell is effectively protected, and after room temperature is placed 36 hours, CIK cell vigor, which remains unchanged, can reach 90%
7th, the present invention inhibits mononuclearcell to CD4+CD25+Treg cell differentiations with PGE2, promotes CIK cell multiplication capacity.The multiplication capacity of cell is further enhanced with IL-4 and GM-CSF simultaneously.In addition, above patent prepares CIK using CD3 monoclonal antibodies coating method, the method can actually improve CIK quantity, but CD3+CD56+ cell proportions are substantially relatively low in final aim cell phenotype, and clinical manifestation is unsatisfactory.Therefore the present invention uses free state CD3 monoclonal antibodies, as a result show, in the presence of PGE2, IL-4 and GM-CSF, CIK cell proliferation times meet or exceed above-mentioned patent method therefor, CD3+CD56+ cell proportions substantially increase simultaneously, and 50% or so is risen to from 30% or so highest.
8th, CIK preparations of the invention when stored between on preparation in more above-mentioned patent be significantly increased.Brought up to 36 hours from 6 hours at room temperature.Reliability is higher in clinical practice, and security obtains effective guarantee.
Fig. 1 is the present invention to adenocarcinoma of lung mdr cell SPC-A1/DTX fragmentation effect figures
In figure, A is blank control, B is the CIK cell that PEG2 is handled, C is the CIK cell that IL-4+GM-CSF is handled, D is the CIK cell that PEG2+IL-4+GM-CSF is handled, three pillars represent 1 ︰ 5,1 ︰ 10 and 1 ︰, 20 3 kinds of ratios in the CIK cell that E is handled for sodium selenite+PEG2+IL-4+GM-CSF, figure.What ordinate was represented is killing rate（%）.
Fig. 2 is the increment situation comparison diagram of cell under the conditions of different disposal
In figure, A is blank control, and B is the CIK cell that PEG2 is handled, and C is the CIK cell that IL-4+GM-CSF is handled, and D is the CIK cell that PEG2+IL-4+GM-CSF is handled, and E is the CIK cell that sodium selenite+PEG2+IL-4+GM-CSF is handled.
Embodiment
In order to preferably explain the present invention, below in conjunction with the specific embodiment main contents that the present invention is furture elucidated, but present disclosure is not limited solely to following examples.
Embodiment 1
The preparation method of one enhanced CIK cell, comprises the following steps：
1）Patient's anticoagulation is gathered under aseptic condition, 1500rpm/min is centrifuged 6 minutes and is drawn upper plasma, 56 DEG C inactivation 30 minutes after centrifuge it is standby, precipitated with haemocyte precipitation volume identical compound normal saline dilution haemocyte, proportion is added in centrifuge tube for 1.077 human lymphocyte separating liquid and diluted blood in 1 ︰ 1.5 ratio, and 2000rpm/min is centrifuged 15 minutes, the leukocytic cream in the middle of careful extraction, cleaned twice with compound physiological saline, PMNC is obtained after low-speed centrifugal（PBMC）；
2）The isolated PMNC from the peripheral blood of collection patient；It is placed in blood-free medium and cultivates, obtains 1 × 106Individual/ml nucleus, adds IFN-γ, phytohemagglutin phytolectin and PGE2 and is transferred in blake bottle or culture bag, is 37 DEG C, 5%CO in temperature2, cultivate 24h under the conditions of saturated humidity；Wherein：The concentration of the IFN-γ：2000IU/ml, PHA concentration：100ng/ml, PGE2 concentration：50μg/ml；
3）Then CD3 monoclonal antibodies are added, IL-2, IL-1 α, IL-4 and GM-CSF continue to cultivate；The concentration of CD3 monoclonal antibodies：20 μ g/ml, IL-2 concentration：1000IU/ml, IL-1 α concentration：50ng/ml；IL-4 concentration：100ng/ml；GM-CSF concentration is 2000U/ml；
4）After culture 3~5, addition contains IL-2 serum free mediums, and control cell density is 1 × 106/ ml, wherein：The concentration of the IL-2：1000IU/ml；
5）Beginning in 7th day was counted every 2~3 days cells to culture, and added the serum free medium containing IL-2 and sodium selenite according to cell concentration, after continuously cultivating the 14th~21 day, and it is 1~6 × 10 to be collected by centrifugation and obtain cell density6Individual/ml enhanced CIK cell, wherein：The concentration of the IL-2 is 1000IU/ml, and the concentration of sodium selenite is 100 μ g/L.
The nature examination of two CIK cells
1st, the morphology of CIK cell, cell viability and immunophenotype detection
Add after PHA, IFN-γ, most cells are still in suspended state；Cell volume increases after 3 days, and cell gradually assembles agglomerating, and cell is bright, and kytoplasm enriches；The starts for 6-7 days, and cell starts a large amount of propagation, and cellular morphology is various, cell mass showed increased；
The μ l of cell 100 of culture 13 days are taken, 100 μ l0.4% Trypan Blue liquid are added, living cells is not colored, and dead cell is dyed to blueness.Cell viability prepared by the present invention is more than 95%.
2nd, CIK cell purity, proliferative ability and immunophenotype detection
Culture 0,4,7,10 days 100 μ l, concentration about 10 are taken respectively6/ ml cell, is separately added under the detection anti-human μ l of CD3-PerCP, CD4-FITC, CD8-PE, CD56-APC antibody 10 of mouse, room temperature environment, lucifuge is incubated 30min.Washed twice afterwards with PBS, carry out FCM analysis.
Nearly 800 times of CIK cell amplification in foreign cell group manufactured in the present embodiment, CD3+ positive T cells ratio is (92.2 ± 1.95) %, CD3+56+ double positive cells from initial (2.0 ± 0.5) % increase to (42.8 ± 3.5) %, CD3+56+ cell culture reach peak value within the 10th day.
3rd, CIK cell is detected to adenocarcinoma of lung mdr cell SPC-A1/DTX lethal effect
Adenocarcinoma of lung mdr cell SPC-A1/DTX is inoculated with 96 orifice plates or 16 orifice plates and is cultivated 24 hours, afterwards enhanced CIK cell prepared by the present invention is added in the effect ︰ 5 of target ratio 1,1 ︰ 10 and 1 ︰ 20 ratio, co-incubation adds the 5mg/ml of 10 μ l Fresh MTT after 24 hours, after co-incubation 4hr, supernatant is abandoned in centrifugation suction, per hole add 100 μ l DMSO vibration dissolving 10 minutes, with enzyme mark detector at 570nm mensuration absorbance A values.
As Fig. 1 shows that CIK cell manufactured in the present embodiment is 6 times of cell prepared by conventional method to adenocarcinoma of lung mdr cell SPC-A1/DTX killing activity, it was demonstrated that IL-2 combinations PHA, PGE2, sodium selenite have synergy to the GVT for strengthening CIK cell.
4th, the increment situation of cell is contrasted under the conditions of different disposal
Process step and method are as follows：
1）CIK progenitor cells are respectively placed in containing B (500ug/ml PGE2), C（100ng/ml IL-4、2000IU/ml GM-CSF）、D(500ug/ml PGE2、100ng/ml IL-4、2000IU/ml GM-CSF)、E（Sodium selenite, 500ug/ml PGE2,100ng/ml IL-4,2000IU/ml GM-CSF）Cell culture fluid in cultivate 24 hours：
2）Move in the blake bottle containing 20ug/ml CD3 monoclonal antibodies, add 2000IU/mlIFN- γ and continue to cultivate, centre carries out fluid infusion, continuous culture obtained required CIK cell to 7-14 days.
As shown in Fig. 2 with the nutrient solution containing D, E, the cell concentration turned out is significantly larger than the cell total amount that other method is turned out.
Embodiment 2
The preparation method of enhanced CIK cell, comprises the following steps：
1）Patient's anticoagulation is gathered under aseptic condition, 1500rpm/min is centrifuged 6 minutes and is drawn upper plasma, 56 DEG C inactivation 30 minutes after centrifuge it is standby, precipitated with haemocyte precipitation volume identical compound normal saline dilution haemocyte, proportion is added in centrifuge tube for 1.077 human lymphocyte separating liquid and diluted blood in 1 ︰ 1.5 ratio, and 2000rpm/min is centrifuged 15 minutes, the leukocytic cream in the middle of careful extraction, cleaned twice with compound physiological saline, PMNC is obtained after low-speed centrifugal（PBMC）；
2）The isolated PMNC from the peripheral blood of collection patient；It is placed in blood-free medium and cultivates, obtains 1 × 106Individual/ml nucleus, adds IFN-γ, phytohemagglutin phytolectin and PGE2 and is transferred in blake bottle or culture bag, is 37 DEG C, 5%CO in temperature2, cultivate 24h under the conditions of saturated humidity；Wherein：The concentration of the IFN-γ：100IU/ml, PHA concentration：50 μ g/ml, PGE2 concentration：1ng/ml；
3）Then CD3 monoclonal antibodies are added, IL-2, IL-1 α, IL-4 and GM-CSF continue to cultivate；The concentration of CD3 monoclonal antibodies：1ng/ml, IL-2 concentration：10IU/ml, IL-1 α concentration：1ng/ml；IL-4 concentration：1ng/ml；GM-CSF concentration is 100U/ml；
4）After culture 3~5, addition contains IL-2 serum free mediums, and control cell density is 1 × 106/ ml, wherein：The concentration of the IL-2：10IU/ml；
5）Beginning in 7th day was counted every 2~3 days cells to culture, and added the serum free medium containing IL-2 and sodium selenite according to cell concentration, after continuously cultivating the 14th~21 day, and it is 1~6 × 10 to be collected by centrifugation and obtain cell density8Individual/ml enhanced CIK cell, wherein：The concentration of the IL-2 is 10IU/ml, and the concentration of sodium selenite is 1 μ g/L.
Embodiment 3
The preparation method of enhanced CIK cell, comprises the following steps：
1）Patient's anticoagulation is gathered under aseptic condition, 1500rpm/min is centrifuged 6 minutes and is drawn upper plasma, 56 DEG C inactivation 30 minutes after centrifuge it is standby, precipitated with haemocyte precipitation volume identical compound normal saline dilution haemocyte, proportion is added in centrifuge tube for 1.077 human lymphocyte separating liquid and diluted blood in 1 ︰ 1.5 ratio, and 2000rpm/min is centrifuged 15 minutes, the leukocytic cream in the middle of careful extraction, cleaned twice with compound physiological saline, PMNC is obtained after low-speed centrifugal（PBMC）；
2）The isolated PMNC from the peripheral blood of collection patient；It is placed in blood-free medium and cultivates, obtains 1 × 106Individual/ml nucleus, adds IFN-γ, phytohemagglutin phytolectin and PGE2 and is transferred in blake bottle or culture bag, is 37 DEG C, 5%CO in temperature2, cultivate 24h under the conditions of saturated humidity；Wherein：The concentration of the IFN-γ：1000IU/ml, PHA concentration：10ng/ml, PGE2 concentration：20ng/ml；
3）Then CD3 monoclonal antibodies are added, IL-2, IL-1 α, IL-4 and GM-CSF continue to cultivate；The concentration of CD3 monoclonal antibodies：100ng/ml, IL-2 concentration：10000IU/ml, IL-1 α concentration：50μg/ml；IL-4 concentration：1μg/ml；GM-CSF concentration is 10000U/ml；
4）After culture 3~5, addition contains IL-2 serum free mediums, and control cell density is 1 × 106/ ml, wherein：The concentration of the IL-2：10000IU/ml；
5）Beginning in 7th day was counted every 2~3 days cells to culture, and added the serum free medium containing IL-2 and sodium selenite according to cell concentration, after continuously cultivating the 14th~21 day, and it is 1~6 × 10 to be collected by centrifugation and obtain cell density7Individual/ml enhanced CIK cell, wherein：The concentration of the IL-2 is 10000IU/ml, and the concentration of sodium selenite is 1mg/L.
Embodiment 4
Enhanced CIK cell preparation is prepared with embodiment 1
1st, the preparation of composite nutrient-fluid is shown in Table 1
Table 1
2nd, prepared by the collection of enhanced CIK cell and preparation
The enhanced CIK cell of 6 pipe culture 14 days is collected, each pipe 1-2L, 1500rmp centrifugation 7min abandon supernatant, collect precipitation.The composite nutrient-fluid prepared with above-mentioned six embodiments distinguishes resuspended precipitate C IK cells, and 1500rmp centrifugation 7min abandon supernatant, collect precipitation.Repeat above step once.The CIK cell of collection is resuspended in corresponding 150ml compound nutrient liquors, six enhanced CIK cell preparations is thus made, its label is respectively 1,2,3,4,5,6, composite nutrient-fluid prepared by six embodiments of correspondence.
3rd, enhanced CIK cell preparation viability examination
6 cell preparations obtained as above are placed in room temperature, collection 0,3,6,9,12,24,36,48 hours samples detect cell viability using trypan exclusion stain.With common CIK preparations（By CIK cell, 20g/L human serum albumin, 2 × 10 obtained by this law5IU/ml IL-2,9g/L NaCl compositions）Compare.Show that common CIK preparations are placed in vigor after room temperature 6 hours and are remarkably decreased by the result of table 2, and the preparation prepared by the present invention can ensure CIK cell after room temperature is placed 36 hours, vigor, which remains unchanged, is maintained at 90%, is 6 times of conventional formulation, wherein, the effect of embodiment 3 is best.
Table 2
Other unspecified parts are prior art.Although above-described embodiment is made that detailed description to the present invention; but it is only a part of embodiment of the invention; rather than whole embodiments, people can also obtain other embodiment according to the present embodiment under the premise of without creativeness, and these embodiments belong to the scope of the present invention.
Claims (8)
1. a kind of preparation method of enhanced CIK cell, it is characterised in that：Comprise the following steps：
1）The isolated PMNC from the peripheral blood of collection patient；It is placed in blood-free medium and cultivates, obtains concentration 1~3 × 106/ ml nucleus, adds IFN-γ, phytohemagglutin phytolectin and PGE2 and is transferred in blake bottle or culture bag, is 37 DEG C, 5%CO in temperature2, cultivate 24h under the conditions of saturated humidity；Wherein：The concentration of the IFN-γ：100~2000u/ml, PHA concentration：10ng~50 μ g/ml, PGE2 concentration：The μ g/ml of 1ng~50；
2）Then CD3 monoclonal antibodies are added, IL-2, IL-1 α, IL-4 and GM-CSF continue to cultivate；The concentration of CD3 monoclonal antibodies：1ng~20 μ g/ml, IL-2 concentration：10~10000IU/ml, IL-1 α concentration：The μ g/ml of 1ng~50；IL-4 concentration：The μ g/ml of 1ng~1；GM-CSF concentration is 100~10000U/ml；
3）After culture 3~5 days, addition contains IL-2 serum free mediums, and control cell density is 1~6 × 106Individual/ml, wherein：The concentration of the IL-2：10~10000IU/ml；
4）Beginning in 7th day was counted every 2~3 days cells to culture, and added the serum free medium containing IL-2 and sodium selenite according to cell concentration, after continuously cultivating the 14th~21 day, is collected by centrifugation and is obtained enhanced CIK cell, wherein：The concentration of the IL-2 is 10~10000IU/ml, and the concentration of sodium selenite is 1 μ g~1mg/L.
2. the preparation method of enhanced CIK cell according to claim 1, it is characterised in that：The cell density of the enhanced CIK cell：1~6 × 106-8Individual/ml.
3. the preparation method of enhanced CIK cell according to claim 2, it is characterised in that：Comprise the following steps：
1）The isolated PMNC from the peripheral blood of collection patient；It is placed in blood-free medium and cultivates, obtains 1 × 106Individual/ml nucleus, adds IFN-γ, phytohemagglutin phytolectin and PGE2 and is transferred in blake bottle or culture bag, is 37 DEG C, 5%CO in temperature2, cultivate 24h under the conditions of saturated humidity；Wherein：The concentration of the IFN-γ：2000IU/ml, PHA concentration：100ng/ml, PGE2 concentration：50μg/ml；
2）Then CD3 monoclonal antibodies are added, IL-2, IL-1 α, IL-4 and GM-CSF continue to cultivate；The concentration of CD3 monoclonal antibodies：20 μ g/ml, IL-2 concentration：1000IU/ml, IL-1 α concentration：50ng/ml；IL-4 concentration：100ng/ml；GM-CSF concentration is 2000U/ml；
3）After culture 3~5, addition contains IL-2 serum free mediums, and control cell density is 1 × 106/ ml, wherein：The concentration of the IL-2：1000IU/ml；
4）Beginning in 7th day was counted every 2~3 days cells to culture, and added the serum free medium containing IL-2 and sodium selenite according to cell concentration, after continuously cultivating the 14th~21 day, is collected by centrifugation and is obtained enhanced CIK cell, wherein：The concentration of the IL-2 is 1000IU/ml, and the concentration of sodium selenite is 100 μ g/L.
4. a kind of enhanced CIK cell preparation, enhanced CIK cell, human serum albumin, IL-2, amino acid, vitamin and the inorganic salts obtained using the preparation method of the enhanced CIK cell described in any one in claims 1 to 3.
5. enhanced CIK cell preparation according to claim 4, it is characterised in that：The human serum albumin concentration：1~300g/L, IL-2 concentration：L~9 × 105~7U/ml, amino acid concentration：50mg~10g/L, vitamine concentration：100ug~50mg/L, inorganic salt concentration：500mg~10g/L.
6. enhanced CIK cell preparation according to claim 5, it is characterised in that：The human serum albumin concentration：50~200g/L, IL-2 concentration：L~9 × 105~6U/ml, amino acid concentration：500mg~5g/L, vitamine concentration：1~30mg/L, inorganic salt concentration：1g~7g/L.
7. enhanced CIK cell preparation according to claim 6, it is characterised in that：The human serum albumin concentration：100g/L, IL-2 concentration：2×106U/ml, amino acid concentration：3g/L, vitamine concentration：15mg/L, inorganic salt concentration：4g/L.
8. the enhanced CIK cell preparations according to any one in claim 4 or 5 or 6 or 7, it is characterised in that：The amino acid is any five kinds or more than five kinds in L-arginine, L-ASPARTIC ACID, Pidolidone, L-Histidine, ILE, L lysine HCL, L-phenylalanine, Serine, L-Trp, Valine, L- asparagines, CYSTINE dihydrochloride, glycine, L- hydroxyprolines, L-Leu, METHIONINE, L-PROLINE, L-threonine and TYR；
The vitamin is any three kinds or more in folic acid, riboflavin, biotin, p-aminobenzoic acid, puridoxine hydrochloride, vitamin B12 and niacinamide；
The inorganic salts be in sodium chloride, potassium chloride, AMSP, calcium nitrate and sodium acid carbonate it is any two or more.
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