CN103937741B - 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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- 230000035945 sensitivity Effects 0.000 description 1
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- 238000001228 spectrum Methods 0.000 description 1
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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 enhancement mode CIK cell preparation method and
Cell preparation.
Background technology
Adoptive cellular immunotherapy is referred to by being transfused itself or homospecificity, non-specific antineoplastic immune effect
The treatment method of cell, direct killing tumour cell or virus.Oneself enters clinical practice to adoptive cellular immunotherapy, and becomes
The fourth-largest tumor therapeuticing method after operation, radiotherapy, chemotherapy.The species of such immunocyte includes Lymphokine
Cell (LAK), tumor-infiltrating lymphocytes (TIL), cellulotoxic lymphocyte (CTL) etc. are killed, in numerous immunocytes,
CIK cell is high due to killing tumor activity, kills knurl spectrum extensively, to the same sensitivity of various mdr cells and to normal marrow hemopoiesis premise
The good characteristics such as cytotoxicity is less, are widely used in clinic.
Compared with other immunocytes, CIK cell be oneself know to the most strong effector cell of tumor cytotoxicity activity it
One.It is the synergy by cytokine profiles, a group foreign cell come by PMNC culture.
The quantity and cytotoxic activity of CIK cell directly determines its effect in clinical treatment.NKT cells are CIK foreign cells
The cell of most cytotoxic activity in group, its cell surface expresses two kinds of membrane protein molecules of CD3 and CD56 simultaneously（That is CD3+With
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.So
And, NKT cell contents are extremely low in the peripheral blood of people, and 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, cannot by the immunocyte of itself
The effect for killing tumour is reached, the absolute quantity of this kind of cell by vitro culture, can only be improved.
CIK cell can be by external evoked and breed in a large number, and general CIK preparation methods are that detached peripheral blood is single
After nucleus (PBMC) is processed 24 hours with IFN-γ, CD3mAb, the IL-1 α and IL-2 factor is added to carry out stimulation induction, finally
A number of CIK cell is obtained, but the increment multiple and cytotoxic activity of the final CIK cell for obtaining are all not ideal enough.
The content of the invention
The technical problem to be solved is just to provide a kind of high proliferation power, enhancement mode CIK of high cytotoxic activity
Cell preparation preparation method and cell preparation, the CIK preparations when stored between on be significantly increased.At room temperature from 6
Hour brings up to 36 hours.Reliability is higher in clinical practice, and security obtains effective guarantee.
To solve above-mentioned technical problem, a kind of preparation method of enhancement mode CIK cell that the present invention is provided, including following step
Suddenly：
1）From isolated PMNC in the peripheral blood of collection patient；It is placed in blood-free medium and trains
Support, obtain 1~3 × 106The mononuclearcell of individual/ml, adds IFN-γ, phytohemagglutin phytolectin and PGE2 and is transferred to blake bottle
Or be 37 DEG C, 5%CO in temperature in culture bag2, cultivate 24h under the conditions of saturated humidity；
Wherein：The concentration of the IFN-γ：The concentration of 100~2000u/ml, PHA：10ng~50 μ g/ml, PGE2's is dense
Degree：1ng~50 μ g/ml；
2）CD3 monoclonal antibodies are subsequently adding, IL-2, IL-1 α, IL-4 and GM-CSF continue to cultivate；The concentration of CD3 monoclonal antibodies：1ng
~20 μ g/ml, the concentration of IL-2：The concentration of 10~10000IU/ml, IL-1 α：1ng~50 μ g/ml；The concentration of IL-4：1ng~
1μg/ml；The concentration of GM-CSF 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, its
In：The concentration of the IL-2：10~10000IU/ml；
4）Start within 7th day to be counted every 2~3 days cells to cultivating, and IL-2 is contained according to cell concentration addition
With the serum free medium of sodium selenite, after continuously cultivating the 14th~21 day, it is collected by centrifugation and obtains enhancement mode 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 enhancement mode CIK cell：1~6 × 106-8Individual/ml
Preferably, the preparation method of enhancement mode CIK cell, comprises the following steps：
1）From isolated PMNC in the peripheral blood of collection patient；It is placed in blood-free medium and trains
Support, obtain 1 × 106The nucleus of individual/ml, adds IFN-γ, phytohemagglutin phytolectin and PGE2 and is transferred to blake bottle or culture bag
In, it 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,
The concentration of PHA：The concentration of 100ng/ml, PGE2：50μg/ml；
2）CD3 monoclonal antibodies are subsequently adding, IL-2, IL-1 α, IL-4 and GM-CSF continue to cultivate；The concentration of CD3 monoclonal antibodies：20μ
The concentration of g/ml, IL-2：The concentration of 1000IU/ml, IL-1 α：50ng/ml；The concentration of IL-4：100ng/ml；GM-CSF's is dense
Spend for 2000U/ml；
3）After culture 3~5, addition contains IL-2 serum free mediums, and control cell density is 1 × 106/ ml, wherein：Institute
State the concentration of IL-2：1000IU/ml；
4）Start within 7th day to be counted every 2~3 days cells to cultivating, and IL-2 is contained according to cell concentration addition
With the serum free medium of sodium selenite, after continuously cultivating the 14th~21 day, it is collected by centrifugation and obtains cell density for 1~6 × 106
The enhancement mode CIK cell enhancement mode CIK cell of individual/ml, wherein：The concentration of the IL-2 be 1000IU/ml, sodium selenite it is dense
Spend for 100 μ g/L.
Present invention also offers a kind of enhancement mode CIK cell preparation, including using any one institute in claim 1~3
Enhancement mode CIK cell, human serum albumin, IL-2, amino acid, vitamin that the preparation method of the enhancement mode CIK cell stated is obtained
And inorganic salts.
Further, the human serum albumin concentration：The concentration of 1~300g/L, IL-2：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：The concentration of 50~200g/L, IL-2：L~9 × 105~6U/ml, ammonia
Base acid concentration：500mg~5g/L, vitamine concentration：1~30mg/L, inorganic salt concentration：1g~7g/L.
Yet further, the human serum albumin concentration：The concentration of 100g/L, IL-2：2×106U/ml, amino acid are dense
Degree：3g/L, vitamine concentration：15mg/L, inorganic salt concentration：4g/L.
Yet further, the amino acid is L-arginine, L-ASPARTIC ACID, Pidolidone, L-Histidine, the different bright ammonia of L-
Acid, L lysine HCL, L-phenylalanine, Serine, L-Trp, Valine, L- asparagines, CYSTINE two
Appoint in hydrochloride, glycine, L- hydroxyprolines, L-Leu, METHIONINE, L-PROLINE, L-threonine and TYR
Five kinds or more than five kinds of meaning；
The vitamin is folic acid, riboflavin, biotin, p-aminobenzoic acid, puridoxine hydrochloride, vitamin B12 and cigarette
Any three kinds or more in acid amides；
The inorganic salts be sodium chloride, potassium chloride, AMSP, calcium nitrate and sodium acid carbonate in any two kinds or
It is two or more.
The beneficial effects of the present invention is：
1st, CIK cell can be by external evoked and breed in a large number, and general CIK preparation methods are by detached peripheral blood list
After individual nucleus (PBMC) is processed 24 hours with IFN-γ, CD3mAb, the IL-1 α and IL-2 factor is added to carry out stimulation induction, most
A number of CIK cell is obtained eventually, but the increment multiple and cytotoxic activity of the final CIK cell for obtaining are all not ideal enough.
2nd, in CIK cell incubation it was found that add IFN-γ to stimulate, PBMC can occur apoptosis, have impact on thin
The amplification of born of the same parents.And PHA is essentially identical to the inducing effect and IFN-γ of PBMC, and with efficient proliferation, can make
For the strong activator of T cell, immunocompetent cell precursor is promoted to convert to immunocompetent cell.Therefore replaced with PHA in the present invention
For part IFN-γ improving the increment multiple of CIK cell.
3rd, the CTLA-4 that CD4+CD25+Treg cells pass through activating T cell, suppresses the immunologic function of the T cell of activation.And
PGE2 can suppress mononuclearcell to CD4+CD25+Treg cell differentiations.Therefore activation CIK is strengthened in the present invention with PGE2
The effect of cell.
4th, IL-4 and GM-CSF have synergy, and while PBMC multiplication capacities are strengthened Neutrophil-mediated can be strengthened
Phagocytosis, killing activity.
5th, sodium selenite can improve the toxicity of CIK cell, therefore in the present invention, we add Asia in CIK cell culture
Sodium selenate, 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
It is not appropriate for CIK cell to maintain vigour, preparation must feed back in patient's body after preparing in 6 hours, otherwise CIK cell is lived
Power is significantly reduced, and loses therapeutic action.After this method is adopted containing several amino acids, the nutrient solution of inorganic salts, CIK cell is obtained
Effective protection, after room temperature is placed 36 hours, CIK cell vigor can still reach 90%
7th, the present invention inhibits mononuclearcell to CD4+CD25+Treg cell differentiations with PGE2, promotes CIK cell increasing
Grow ability.Further enhance the multiplication capacity of cell with IL-4 and GM-CSF simultaneously.In addition, above patent adopts CD3 monoclonal antibodies
Coating method prepares CIK, and the method can actually improve the quantity of CIK, but the CD3+CD56+ in final aim cell phenotype
Cell proportion is substantially low, and clinical manifestation is unsatisfactory.Therefore the present invention adopts free state CD3 monoclonal antibodies, as a result shows,
In the presence of PGE2, IL-4 and GM-CSF, CIK cell proliferation times meet or exceed above-mentioned patent method therefor, while CD3
+ CD56+ cell proportions substantially increase, and from 30% or so highest 50% or so is risen to.
8th, CIK preparations of the invention when stored between on preparation in more above-mentioned patent be significantly increased.In room ambient conditions
Under brought up to 36 hours from 6 hours.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, and B is the CIK cell that PEG2 is processed, and C is the CIK cell that IL-4+GM-CSF is processed, and D is
The CIK cell of PEG2+IL-4+GM-CSF process, E is the CIK cell that sodium selenite+PEG2+IL-4+GM-CSF is processed, in figure
Three pillars represent 1 ︰ 5,1 ︰ 10 and 1 ︰, 20 3 kinds of ratios.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, and B is the CIK cell that PEG2 is processed, and C is the CIK cell that IL-4+GM-CSF is processed, and D is
The CIK cell of PEG2+IL-4+GM-CSF process, E is the CIK cell that sodium selenite+PEG2+IL-4+GM-CSF is processed.
Specific embodiment
In order to preferably explain the present invention, the main contents of the present invention are further elucidated below in conjunction with specific embodiment, but
Present disclosure is not limited solely to following examples.
Embodiment 1
The preparation method of one enhancement mode CIK cell, comprises the following steps：
1）Patient's anticoagulation is gathered under aseptic condition, 1500rpm/min is centrifuged and draws within 6 minutes upper plasma, 56 DEG C of inactivations
Be centrifuged after 30 minutes it is standby, with haemocyte precipitation volume identical compound normal saline dilution haemocyte precipitation, proportion is
1.077 human lymphocyte separating liquid and diluted blood are added in centrifuge tube in the ratio of 1 ︰ 1.5, and 2000rpm/min is centrifuged 15 points
Clock, it is careful to extract middle leukocytic cream, cleaned twice with compound physiological saline, the single core of peripheral blood is obtained after low-speed centrifugal thin
Born of the same parents（PBMC）；
2）From isolated PMNC in the peripheral blood of collection patient；It is placed in blood-free medium and trains
Support, obtain 1 × 106The nucleus of individual/ml, adds IFN-γ, phytohemagglutin phytolectin and PGE2 and is transferred to blake bottle or culture bag
In, it 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,
The concentration of PHA：The concentration of 100ng/ml, PGE2：50μg/ml；
3）CD3 monoclonal antibodies are subsequently adding, IL-2, IL-1 α, IL-4 and GM-CSF continue to cultivate；The concentration of CD3 monoclonal antibodies：20μ
The concentration of g/ml, IL-2：The concentration of 1000IU/ml, IL-1 α：50ng/ml；The concentration of IL-4：100ng/ml；GM-CSF's is dense
Spend for 2000U/ml；
4）After culture 3~5, addition contains IL-2 serum free mediums, and control cell density is 1 × 106/ ml, wherein：Institute
State the concentration of IL-2：1000IU/ml；
5）Start within 7th day to be counted every 2~3 days cells to cultivating, and IL-2 is contained according to cell concentration addition
With the serum free medium of sodium selenite, after continuously cultivating the 14th~21 day, it is collected by centrifugation and obtains cell density for 1~6 × 106
The enhancement mode CIK cell of individual/ml, 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
After adding PHA, IFN-γ, most cells are in still suspended state；Cell volume increase after 3 days, cell gradually gathers
Collection is 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, and cell mass is obvious
Increase；
The culture μ l of cell 100 of 13 days are taken, adds 100 μ l0.4% Trypan Blue liquid, living cells not to be colored, dead cell
It is dyed to blueness.Cell viability prepared by the present invention is more than 95%.
2nd, CIK cell purity, proliferative ability and immunophenotype detection
0,4,7,10 days 100 μ l of culture, concentration about 10 are taken respectively6The cell of/ml, is separately added into the detection anti-human CD3- of mouse
The μ l of PerCP, CD4-FITC, CD8-PE, CD56-APC antibody 10, under room temperature environment, lucifuge incubation 30min.Washed with PBS afterwards
Twice, FCM analysis are carried out.
CIK cell amplification is nearly 800 times in foreign cell group manufactured in the present embodiment, and CD3+ positive T cells ratio is (92.2
± 1.95) %, CD3+56+ double positive cells increase to (42.8 ± 3.5) % from initial (2.0 ± 0.5) %, and CD3+56+ cells exist
Cultivate the 10th day and reach peak value.
3rd, CIK cell is detected to the lethal effect of adenocarcinoma of lung mdr cell SPC-A1/DTX
Adenocarcinoma of lung mdr cell SPC-A1/DTX is inoculated with 96 orifice plates or 16 orifice plates and is cultivated 24 hours, afterwards by effect target ratio
The ratio of the ︰ 5 of example 1,1 ︰ 10 and 1 ︰ 20 adds enhancement mode CIK cell prepared by the present invention, co-incubation that 10 μ l are added after 24 hours
The MTT of freshly prepared 5mg/ml, after co-incubation 4hr, supernatant is abandoned in centrifugation suction, adds the DMSO vibrations of 100 μ l molten per hole
Solution 10 minutes, with enzyme mark detector mensuration absorbance A value at 570nm.
As Fig. 1 shows that CIK cell manufactured in the present embodiment is to the killing activity of adenocarcinoma of lung mdr cell SPC-A1/DTX
6 times of cell prepared by conventional method, it was demonstrated that IL-2 combination PHA, the antitumor effect of PGE2, sodium selenite to enhancing CIK cell
There should be synergy.
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）In moving to the blake bottle containing 20ug/ml CD3 monoclonal antibodies, 2000IU/mlIFN- γ are added to continue to cultivate, it is middle
Fluid infusion is carried out, is continuously cultivated to 7-14 days and is obtained required CIK cell.
As shown in Fig. 2 with the nutrient solution containing D, E, it is thin that the cell concentration turned out is significantly larger than that additive method turns out
Born of the same parents' total amount.
Embodiment 2
The preparation method of enhancement mode CIK cell, comprises the following steps：
1）Patient's anticoagulation is gathered under aseptic condition, 1500rpm/min is centrifuged and draws within 6 minutes upper plasma, 56 DEG C of inactivations
Be centrifuged after 30 minutes it is standby, with haemocyte precipitation volume identical compound normal saline dilution haemocyte precipitation, proportion is
1.077 human lymphocyte separating liquid and diluted blood are added in centrifuge tube in the ratio of 1 ︰ 1.5, and 2000rpm/min is centrifuged 15 points
Clock, it is careful to extract middle leukocytic cream, cleaned twice with compound physiological saline, the single core of peripheral blood is obtained after low-speed centrifugal thin
Born of the same parents（PBMC）；
2）From isolated PMNC in the peripheral blood of collection patient；It is placed in blood-free medium and trains
Support, obtain 1 × 106The nucleus of individual/ml, adds IFN-γ, phytohemagglutin phytolectin and PGE2 and is transferred to blake bottle or culture bag
In, it 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,
The concentration of PHA：50 μ g/ml, the concentration of PGE2：1ng/ml；
3）CD3 monoclonal antibodies are subsequently adding, IL-2, IL-1 α, IL-4 and GM-CSF continue to cultivate；The concentration of CD3 monoclonal antibodies：1ng/
The concentration of ml, IL-2：The concentration of 10IU/ml, IL-1 α：1ng/ml；The concentration of IL-4：1ng/ml；The concentration of GM-CSF is
100U/ml；
4）After culture 3~5, addition contains IL-2 serum free mediums, and control cell density is 1 × 106/ ml, wherein：Institute
State the concentration of IL-2：10IU/ml；
5）Start within 7th day to be counted every 2~3 days cells to cultivating, and IL-2 is contained according to cell concentration addition
With the serum free medium of sodium selenite, after continuously cultivating the 14th~21 day, it is collected by centrifugation and obtains cell density for 1~6 × 108
The enhancement mode CIK cell of individual/ml, 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 enhancement mode CIK cell, comprises the following steps：
1）Patient's anticoagulation is gathered under aseptic condition, 1500rpm/min is centrifuged and draws within 6 minutes upper plasma, 56 DEG C of inactivations
Be centrifuged after 30 minutes it is standby, with haemocyte precipitation volume identical compound normal saline dilution haemocyte precipitation, proportion is
1.077 human lymphocyte separating liquid and diluted blood are added in centrifuge tube in the ratio of 1 ︰ 1.5, and 2000rpm/min is centrifuged 15 points
Clock, it is careful to extract middle leukocytic cream, cleaned twice with compound physiological saline, the single core of peripheral blood is obtained after low-speed centrifugal thin
Born of the same parents（PBMC）；
2）From isolated PMNC in the peripheral blood of collection patient；It is placed in blood-free medium and trains
Support, obtain 1 × 106The nucleus of individual/ml, adds IFN-γ, phytohemagglutin phytolectin and PGE2 and is transferred to blake bottle or culture bag
In, it 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,
The concentration of PHA：The concentration of 10ng/ml, PGE2：20ng/ml；
3）CD3 monoclonal antibodies are subsequently adding, IL-2, IL-1 α, IL-4 and GM-CSF continue to cultivate；The concentration of CD3 monoclonal antibodies：
The concentration of 100ng/ml, IL-2：The concentration of 10000IU/ml, IL-1 α：50μg/ml；The concentration of IL-4：1μg/ml；GM-CSF's
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：Institute
State the concentration of IL-2：10000IU/ml；
5）Start within 7th day to be counted every 2~3 days cells to cultivating, and IL-2 is contained according to cell concentration addition
With the serum free medium of sodium selenite, after continuously cultivating the 14th~21 day, it is collected by centrifugation and obtains cell density for 1~6 × 107
The enhancement mode CIK cell of individual/ml, wherein：The concentration of the IL-2 is 10000IU/ml, and the concentration of sodium selenite is 1mg/L.
Embodiment 4
With the preparation enhancement mode CIK cell preparation of embodiment 1
1st, the preparation of composite nutrient-fluid is shown in Table 1
Table 1
2nd, prepared by the collection of enhancement mode CIK cell and preparation
The 6 pipe culture enhancement mode CIK cell of 14 days is collected, each pipe 1-2L, 1500rmp centrifugation 7min abandons supernatant, collects
Precipitation.The composite nutrient-fluid prepared with above-mentioned six embodiments distinguishes resuspended precipitate C IK cell, and 1500rmp centrifugation 7min are abandoned
Clearly, precipitation is collected.Repeat above step once.In corresponding 150ml compound nutrient liquors, thus the CIK cell of collection is resuspended in
Six enhancement mode CIK cell preparations are obtained, its label is respectively 1,2,3,4,5,6, compound battalion prepared by six embodiments of correspondence
Nutrient solution.
3rd, enhancement mode CIK cell preparation viability examination
6 cell preparations obtained as above are placed in into room temperature, 0,3,6,9,12,24,36,48 hours samples are gathered, using platform
Expect that indigo plant refuses dye method detection cell viability.With common CIK preparations（By this law gained CIK cell, the human serum albumin of 20g/L, 2 ×
105The NaCl compositions of IL-2,9g/L of IU/ml）Compare.After showing that common CIK preparations are placed in room temperature 6 hours by the result of table 2
Vigor is remarkably decreased, and the preparation prepared by the present invention can ensure that CIK cell after room temperature is placed 36 hours, and vigor is still protected
Hold 90%, be 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 to the present invention retouching in detail
State, but it is only a part of embodiment of the invention, rather than whole embodiments, people can with according to the present embodiment without
Other embodiment is obtained under the premise of creativeness, these embodiments belong to the scope of the present invention.
Claims (7)
1. a kind of preparation method of enhancement mode CIK cell, it is characterised in that：Comprise the following steps：
1) from isolated PMNC in the peripheral blood of collection patient；It is placed in blood-free medium and cultivates,
Obtain 1 × 106The nucleus of individual/ml, adds IFN-γ, phytohemagglutin phytolectin and PGE2 and is transferred in blake bottle or culture bag,
It 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, plants
The concentration of thing hemagglutinin：The concentration of 100ng/ml, PGE2：50μg/ml；
2) CD3 monoclonal antibodies are subsequently adding, IL-2, IL-1 α, IL-4 and GM-CSF continue to cultivate；The concentration of CD3 monoclonal antibodies：20 μ g/ml,
The concentration of IL-2：The concentration of 1000IU/ml, IL-1 α：50ng/ml；The concentration of IL-4：100ng/ml；The concentration of GM-CSF is
2000U/ml；
3) after cultivating 3~5 days, addition contains IL-2 serum free mediums, and control cell density is 1 × 106Individual/ml, wherein：Institute
State the concentration of IL-2：1000IU/ml；
4) start within the 7th day to be counted every 2~3 days cells to cultivating, and according to cell concentration addition containing IL-2 and Asia
The serum free medium of sodium selenate, after continuously cultivating the 14th~21 day, is collected by centrifugation and obtains enhancement mode CIK cell, wherein：It is described
The concentration of IL-2 is 1000IU/ml, and the concentration of sodium selenite is 100 μ g/L.
2. a kind of enhancement mode CIK cell preparation, including being obtained using the preparation method of the enhancement mode CIK cell described in claim 1
Enhancement mode CIK cell, the human serum albumin for arriving：1g/L、IL-2：9×107U/ml, L-arginine：1000mg/L, L-Histidine：
500mg/L, L lysine HCL：2000mg/L, L-Trp：700mg/L, L- asparagine：800mg/L, glycine：
3000mg/L, METHIONINE：1000mg/L, L-threonine：1000mg/L, folic acid：0.01mg/L, biotin：0.02mg/L、
Niacinamide：0.02mg/L, sodium chloride：30mg/L and sodium acid carbonate：20mg/L is constituted.
3. a kind of enhancement mode CIK cell preparation, including being obtained using the preparation method of the enhancement mode CIK cell described in claim 1
Enhancement mode CIK cell, the human serum albumin for arriving：300g/L、IL-2：1×105U/ml, L-ASPARTIC ACID：5mg/L, L- paddy ammonia
Acid：8mg/L, L-phenylalanine：10mg/L, Serine：15mg/L, Valine：5mg/L, CYSTINE dihydrochloride：
10mg/L, L- hydroxyproline：4mg/L, L-Leu：2mg/L, L-PROLINE：1mg/L, riboflavin：10mg/L, p-aminophenyl
Formic acid：20mg/L, puridoxine hydrochloride：20mg/L, potassium chloride：5000mg/L, AMSP：1000mg/L, calcium nitrate：
3500mg/L and sodium acid carbonate：500mg/L is constituted.
4. a kind of enhancement mode CIK cell preparation, including being obtained using the preparation method of the enhancement mode CIK cell described in claim 1
Enhancement mode CIK cell, the human serum albumin for arriving：20g/L、IL-2：2×106U/ml, L-arginine：280mg/L, L- door winter ammonia
Acid：20mg/L, Pidolidone：20mg/L, L-Histidine：15mg/L, ILE：50mg/L, L lysine HCL：
40mg/L, L-phenylalanine：15mg/L, Serine：30mg/L, L-Trp：5mg/L, Valine：20mg/L, L- door
Winter acid amides：50mg/L, CYSTINE dihydrochloride：65mg/L, glycine：10mg/L, L- hydroxyproline：The bright ammonia of 20mg/L, L-
Acid：50mg/L, METHIONINE 15mg/L, L-PROLINE：20mg/L, L-threonine：20mg/L, TYR：20mg/L, leaf
Acid：1mg/L, riboflavin：0.2mg/L, biotin：0.2mg/L, p-aminobenzoic acid：1mg/L, puridoxine hydrochloride：1mg/L, dimension
Raw element B12：0.005mg/L, niacinamide：1mg/L, sodium chloride：6000mg/L, potassium chloride：400mg/L, AMSP：
670mg/L, calcium nitrate：100mg/L and sodium acid carbonate：2000mg/L is constituted.
5. a kind of enhancement mode CIK cell preparation, including being obtained using the preparation method of the enhancement mode CIK cell described in claim 1
Enhancement mode CIK cell, the human serum albumin for arriving：200g/L、IL-2：5×106U/ml, L-arginine：200mg/L, L- group ammonia
Acid：50mg/L, L-phenylalanine：10mg/L, L-Trp：5mg/L, L- asparagine：45mg/L, L- hydroxyproline：50mg/
L, L-Leu：30mg/L, L-PROLINE：50mg/L, L-threonine：30mg/L, TYR：40mg/L, folic acid：0.2mg/
L, p-aminobenzoic acid：0.3mg/L, vitamin B12：0.2mg/L, niacinamide：0.3mg/L, sodium chloride：It is 2000mg/L, anhydrous
Sodium dihydrogen phosphate：3000mg/L and sodium acid carbonate：2000mg/L is constituted.
6. a kind of enhancement mode CIK cell preparation, including being obtained using the preparation method of the enhancement mode CIK cell described in claim 1
Enhancement mode CIK cell, the human serum albumin for arriving：50g/L、IL-2：2×106U/ml, L-arginine：2000mg/L, L- door winter ammonia
Acid：20mg/L, Pidolidone：60mg/L, ILE：20mg/L, Serine：900mg/L, Valine：500mg/
L, CYSTINE dihydrochloride：700mg/L, glycine：400mg/L, L-Leu：200mg/L, METHIONINE：50mg/L、
L-PROLINE：20mg/L, L-threonine：100mg/L, TYR：30mg/L, folic acid：10mg/L, riboflavin：20mg/L, life
Thing element：5mg/L, p-aminobenzoic acid：5mg/L, puridoxine hydrochloride：2mg/L, vitamin B12：4mg/L, niacinamide：4mg/L、
Sodium chloride：4000mg/L, AMSP：500mg/L, calcium nitrate：500mg/L and sodium acid carbonate：2000mg/L is constituted.
7. a kind of enhancement mode CIK cell preparation, including being obtained using the preparation method of the enhancement mode CIK cell described in claim 1
Enhancement mode CIK cell, the human serum albumin for arriving：100g/L、IL-2：2×106U/ml, L-ASPARTIC ACID：1000mg/L, L- group
Propylhomoserin：500mg/L, L lysine HCL：500mg/L, Serine：300mg/L, Valine：50mg/L, CYSTINE
Dihydrochloride：50mg/L, L- hydroxyproline：600mg/L, METHIONINE：1000mg/L, folic acid：1mg/L, riboflavin：3mg/
L, biotin：4mg/L, p-aminobenzoic acid：10mg/L, vitamin B12：2mg/L, potassium chloride：3000mg/L and calcium nitrate：
2000mg/L is constituted.
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