CN105505871A - Method for effectively amplifying CIK cells and improving specific tumor killing capability of CIK cells - Google Patents
Method for effectively amplifying CIK cells and improving specific tumor killing capability of CIK cells Download PDFInfo
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Abstract
The invention provides a method for effectively amplifying cytokine-induced killer (CIK) cells and improving specific tumor killing capability of the CIK cells. The method comprises the following steps: separating to obtain mononuclear cells; transferring the obtained mononuclear cells to a culture flask; adding CD3mAb with the final concentration of 400-600 ng/ml, CD28mAb with the final concentration of 1400-1600 ng/ml, IFN-gamma with the final concentration of 4000-4800 IU/ml and IL-2 with the final concentration of 4000-4800 IU/ml, IL-15 with the final concentration of 140-160 ng/ml; on the fourth day, replenishing fresh culture solution including 600-1000 IU/ml of IL-2 and 20-30 ng/ml of IL-15; on the sixth day, culturing the cells in separated flasks; on the eighth and ninth days, respectively transferring cells of the flask to a cell culture bag of 1.8 L, and replenishing fresh culture solution including 600-1000 IU/ml of IL-2 and 20-30 ng/ml of IL-15; at the eleventh day, culturing the cells in separated bags, and replenishing fresh culture solution including 600-1000 IU/ml of IL-2 and 20-30 ng/ml IL-15; continuously inducing and amplifying till the cells are obtained from the fifteenth day to the twenty-first day; incubating the obtained cells for 30-40 minutes at the room temperature by utilizing anti-EGFR and anti-CD3 bifunctional antibodies, washing the incubated cells in normal saline, and collecting the cells, thereby obtaining specific tumor-killing CIK cell preparations.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of effective amplification CIK cell and improve the method that its specificity kills knurl.
Background technology
Malignant tumour is No. second killer of the harm humans health after cardiovascular disorder, and in recent years, the incidence of tumour and mortality ratio are in the trend risen year by year, and tumor invasion becomes rejuvenation trend.Along with the fast development of the subject such as molecular biology and immunology, the biological immune of tumour treats the 4th kind of therapy become after operation, radiotherapy, chemotherapy.Wherein, tumour adoptive immunity cell therapy becomes the focus of current research, it refers to and is fed back in patient body by the immunocyte with anti-tumor activity of people's amplification in vitro, thus direct or indirect excitating organism immunne response is with a kind of cell therapy of killing tumor cell.It has the advantages such as individuality, security, targeting and high efficiency.
Cytokine-induced killer cell sexual cell (Cytokine-InducedKillercells, CIK) one in tumour adoptive immunity cell therapy is belonged to, by the SchmidtWolf of Stanford Univ USA in reported first in 1991, their finder's peripheral blood mononuclear cell in cytokine profiles (as IFN-γ, AntiCD3 McAb McAb, IL-1 α and IL-2) act on for some time after, be directed induction and breed to become in a large number and can express CD3 and CD56 two kinds of membrane protein molecules simultaneously, and with the restricted foreign cell group killing knurl advantage of non-MHC of the powerful anti-tumor activity of T lymphocyte and NK cell.CIK cell have proliferation rates in vitro fast, kill tumor activity high, kill that knurl spectrum is wide, untoward reaction is few, to multidrug resistant tumour cell same responsive, can the features such as patient immune function be improved, be the ideal effector cell of a class in clinical application, be considered to the hope of adoptive immunotherapy.
But this type of effector cell quantity in people's normal peripheral blood is few, only accounts for 1% ~ 5%.At present, though people obtain the CIK cell of some amount by the CIK preparation method of routine, cytotoxicity and the proliferation times of the CIK cell obtained are all not ideal enough.As can be seen here, how obtaining the effector cell that quantity is enough, cytotoxicity is strong is the necessary requirement ensureing result for the treatment of.In addition, a feature of CIK cell kills knurl spectrum extensively, mean that its specificity kills knurl ability poor, with regard to certain specific tumour, as EGF-R ELISA (EGFR) positive tumor, the ability how improving its specific killing EGFR positive tumor cell is that lifting CIK cell result for the treatment of institute must not another realistic problem irrespective.The maintenance of EGFR to the growth of tumour, development and tumor stem cell has very important effect, and there is process LAN or unconventionality expression in multiple solid tumor, in this patent research, therefore can be used as an important target.
Summary of the invention
The object of the present invention is to provide a kind of effective amplification CIK and improve the method that its specificity kills knurl ability, the proliferation times solving the CIK cell obtained at present is not ideal enough; Need in CIK Induction Process to continue to supplement various cytokine, comparatively loaded down with trivial details; The feature that CIK cell wide spectrum the kills knurl shortcoming such as knurl ability is weak that causes its specificity to be killed.
For achieving the above object, the present invention adopts following technical scheme:
Be separated with Ficoll lymphocyte separation medium and obtain mononuclearcell, gone in T175 culturing bottle, add CD3mAb, CD28mAb, IFN-γ, IL-2, IL-15, within the 4th day, add IL-2 and IL-15, within the 6th day, sub-bottle is cultivated, 8th, within 9 days, respectively the cell in bottle is gone in 1.8L cell culture bags, add IL-2 and IL-15, within the 11st day, cell splitting is cultivated, adds IL-2 and IL-15, continue induced amplification, harvested cell in 15-21 days.After hatching 30-40 minute under a kind of anti-EGFR of the cell × AntiCD3 McAb bifunctional antibody normal temperature of results, with collecting cell after brine, namely can be made into the CIK cell preparation of specific killing EGFR positive tumor cell.
Aseptic collection Healthy People or peripheral blood in patients 60ml, be transported to laboratory preparation, carry out the separation of PBMC and the vitro culture of CIK in clean area in 2 hours.Whole blood can carry out later separation and cultivate operation after microorganism immunodetection is qualified.
1. monocyte (PBMC) is separated
A () gets 500ul peripheral blood cellanalyzer counting, Activity determination: mixed with physiological saline equal-volume by peripheral blood, add 40mlFicoll lymphocyte separation medium centrifugation monocyte afterwards; Parameter of noncentricity: 1600rpm, 20 degrees Celsius, 20 minutes;
B () gets tunica albuginea, add physiological saline to cumulative volume 80ml, centrifuge washing twice; Parameter of noncentricity: 2000rpm, 20 degrees Celsius, 8 minutes;
C () cell serum-free basic medium re-suspended cell precipitation, utilizes cell counter to carry out cell counting, calculates cell yield.
2.CIK inducing culture
(1) after the PBMC counting be separated, by 8-10 × 10
5individual/ml density is inoculated in T175 culturing bottle, add final concentration 400-600ng/mlCD3mAb, 1400-1600ng/mlCD28mAb, 4000-4800IU/mlIFN-γ, 4000-4800IU/mlIL-2,140-160ng/mlIL-15, supplement serum free medium to 50ml.
(2) the 3rd days, cell counting, added the fresh medium 50ml of 600-1000IU/mlIL-2,20-30ng/mlIL-15.
(3) the 4th days, cell counting, added the fresh medium 50ml of 600-1000IU/mlIL-2,20-30ng/mlIL-15.
Within (4) the 6th days, sub-bottle is cultivated, cell counting, after being mixed by cell suspension in former culturing bottle, 1.4:1 is transferred in two new T175 culturing bottles respectively by volume, be labeled as a.b and c.d respectively, two bottles of fresh mediums adding 600-1000IU/mlIL-2,20-30ng/mlIL-15 are respectively to 240ml;
Pack in (5) the 8th days is cultivated, cell counting, cell suspension in a.b culturing bottle is loaded 1.8L cell culture bags (bag a) in, add the fresh medium of 600-1000IU/mlIL-2,20-30ng/mlIL-15 to final volume 1000ml.
Pack in (6) the 9th days is cultivated, and cell suspension in c.d culturing bottle loads in 1.8L cell culture bags (bag c), adds the fresh medium of 600-1000IU/mlIL-2,20-30ng/mlIL-15 to final volume 1000ml by cell counting.
Within (7) the 11st days, splitting is cultivated, after cell counting, divided in two new culture bag (bag b and bag d) in 1.4:1 ratio respectively by cell suspension in former culture bag (bag a and bag c), four cell culture bags add the fresh medium of 600-1000IU/mlIL-2,20-30ng/mlIL-15 respectively to final volume 1000ml.
(8) the 15th, 17,19,21 days, cell counting, detected cytoactive, harvested cell (bag a, bag b, bag c, bag d).After the cell gathered in the crops hatches 30-40 minute at normal temperatures with anti-EGFR × AntiCD3 McAb bifunctional antibody respectively, for subsequent use with brine cell.
3.CIK cell is to the Cytotoxicity in vitro rate of EGFR positive tumor cell
(1) by anti-EGFR × AntiCD3 McAb bifunctional antibody treatment group and untreated fish group CIK cell and target cell, add 96 well culture plates in 5 ︰ 1,10 ︰ 1,20 ︰ 1, ratio, simultaneously laying effect groups of cells, target cell group and blank group, often group establishes 3 parallel holes, be placed in 37 DEG C, 5%CO
2, incubator is cultivated.
(2), after 20h, 100 μ l supernatants are taken out in every hole, then add 10 μ lCCK8, continue to hatch 4-6h.
(3) on enzyme-linked immunosorbent assay instrument, select 450nm wavelength to survey each hole OD value.
(4) parallel hole mean OD value calculation result:
Killing activity (%)=1-(experimental group OD value one effect group OD value/target cell group OD value) × 100%.
The invention has the advantages that:
Prior art defect: the proliferation times of the final CIK cell obtained is not ideal enough; Need in CIK Induction Process to continue to supplement various cytokine, comparatively loaded down with trivial details; It kills knurl and composes wide feature and it must be caused to lack specificity kill the ability (kill capability for specific tumors is poor) of knurl.
The present invention newly increases IL-15 and CD28mAb two kinds of factors in traditional C IK induction broth, and (under the synergy of CD28mAb, its stimulating activity significantly improves CD3mAb.IL-2 is under the synergy of IL-15, and its stimulating activity also can significantly improve.), and on culture process, design sub-bottle, turn the step such as bag, splitting, efficient amplification CIK cell number.
The present invention is after separation obtains mononuclearcell, disposablely add CD3 monoclonal antibody (CD3mAb), CD28 monoclonal antibody (CD28mAb), interferon-γ (IFN-γ), interleukin-2 (IL-2), interleukin-15 (IL-15), in follow-up culturing process, fluid infusion is except interpolation basic medium, only need add IL-2 and IL-15, save comparatively loaded down with trivial details operation.
The present invention with collecting cell after brine, makes the CIK cell preparation of specific killing EGFR positive tumor cell after the cell that inducing culture 15-21 days gather in the crops is hatched 30-40 minute at normal temperatures with a kind of anti-EGFR × AntiCD3 McAb bifunctional antibody.
CIK cell culture system: serum-free basic medium, CD3 monoclonal antibody (CD3mAb), CD28 monoclonal antibody (CD28mAb), interferon-γ (IFN-γ), interleukin-2 (IL-2), interleukin-15 (IL-15).
CIK cell culture process: after adding the above-mentioned factor first, follow-up fluid infusion only need add serum-free basic medium, IL-2 and IL-15.In addition, culture process designs sub-bottle, to turn the step such as bag, splitting also different from other people.
Improve specificity and kill the method for knurl ability: the cell that inducing culture 15-21 days are gathered in the crops, hatch at normal temperatures with a kind of bi-specific antibody EGFR × CD3 and make final CIK cell preparation, thus improve its specificity and kill knurl ability.
In CIK cell Induction Process, add CD3 monoclonal antibody (CD3mAb), CD28 monoclonal antibody (CD28mAb), interferon-γ (IFN-γ), interleukin-2 (IL-2), interleukin-15 (IL-15), and except IL-2 and IL-15, its complementary divisor only need add first, reduces troublesome operation.Technical process designs sub-bottle, turn the step such as bag, splitting, fairly large expansion CIK culture system, thus reach the object of amplification CIK quantity.Cultivate 15-21 days harvested cells, and hatch 30-40 minute at normal temperatures with a kind of anti-EGFR × AntiCD3 McAb bifunctional antibody, thus preparation has the CIK cell preparation of specific killing EGFR positive tumor cell.The CIK cell finally obtained is carried out flow cyctometry detection and microbiologic inhibition tests.CIK quantity can be increased to 8 × 10 by CIK cultural method provided by the invention within the time in 2-3 week
9above, Cell viability more than 98%, wherein CD3
+cD56
+two positive cell ratio reaches more than 30%, can up to more than 90% to the Cytotoxicity in vitro rate of EGFR positive tumor cell.
Accompanying drawing explanation
The morphological observation of Fig. 1 CIK cell inducing culture process, the cellular form of figure is respectively CIK cell inducing culture the 2nd day, the 6th day, the 8th day and the 9th day, cell growth state is good as seen from the figure.
Anti-EGFR × AntiCD3 McAb bifunctional antibody CIK and ControlCIK of Fig. 2 difference effect target ratio is to the Cytotoxicity in vitro rate of Hela cell, the lethal effect of CIK to Hela tumour cell as seen from the figure after anti-EGFR × AntiCD3 McAb bifunctional antibody is hatched is not hatched group and is significantly strengthened, when effect target ratio is only 5 ︰ 1, can 60.67% be reached to the kill rate of Hela tumour cell, when imitating target than during for 10:1, can up to 91.26% to the kill rate of Hela tumour cell.
Embodiment
Embodiment 1
Be separated with Ficoll lymphocyte separation medium and obtain mononuclearcell, gone in T175 culturing bottle, add CD3mAb, CD28mAb, IFN-γ, IL-2, IL-15, within 4th day, add IL-2 and IL-15, within the 6th day, sub-bottle is cultivated, and within the 8th, 9 day, goes in 1.8L cell culture bags respectively by the cell in bottle, add IL-2 and IL-15, within 11st day, IL-2 and IL-15 is added in the cultivation of cell splitting, continues induced amplification, harvested cell in 15-21 days.After hatching 30 minutes under a kind of anti-EGFR of the cell × AntiCD3 McAb bifunctional antibody normal temperature of results, with collecting cell after PBS washing, namely can be made into the CIK cell preparation of specific killing EGFR positive tumor cell.
Aseptic collection Healthy People or peripheral blood in patients 60ml, be transported to laboratory preparation, carry out the separation of PBMC and the vitro culture of CIK in clean area in 2 hours.Whole blood can carry out later separation and cultivate operation after microorganism immunodetection is qualified.
1. monocyte (PBMC) is separated
A () gets 500ul peripheral blood cellanalyzer counting, Activity determination: mixed with physiological saline equal-volume by peripheral blood, add 40mlFicoll lymphocyte separation medium centrifugation monocyte afterwards; Parameter of noncentricity: 1600rpm, 20 degrees Celsius, 20 minutes;
B () gets tunica albuginea, add physiological saline to cumulative volume 80ml, centrifuge washing twice; Parameter of noncentricity: 2000rpm, 20 degrees Celsius, 8 minutes;
C () cell serum-free basic medium re-suspended cell precipitation, utilizes cell counter to carry out cell counting, calculates cell yield.
2.CIK inducing culture
(1) after the PBMC counting be separated, by 8 × 10
5individual/ml density is inoculated in T175 culturing bottle, and adding final concentration is 400ng/mlCD3mAb, 1400ng/mlCD28mAb, 4000IU/mlIFN-γ, 4000IU/mlIL-2,140ng/mlIL-15, supplements serum free medium to 50ml.
(2) the 3rd days, cell counting, added the fresh medium 50ml of 600IU/mlIL-2,20ng/mlIL-15.
(3) the 4th days, cell counting, added the fresh medium 50ml of 600IU/mlIL-2,20ng/mlIL-15.
Within (4) the 6th days, sub-bottle is cultivated, cell counting, after being mixed by cell suspension in former culturing bottle, 1.4:1 is transferred in two new T175 culturing bottles respectively by volume, and be labeled as a.b and c.d respectively, two bottles of fresh mediums adding 600IU/mlIL-2,20ng/mlIL-15 are respectively to 240ml;
Pack in (5) the 8th days is cultivated, cell counting, cell suspension in a.b culturing bottle is loaded 1.8L cell culture bags (bag a) in, add the fresh medium of 600IU/mlIL-2,20ng/mlIL-15 to final volume 1000ml.
Pack in (6) the 9th days is cultivated, and cell suspension in c.d culturing bottle loads in 1.8L cell culture bags (bag c), adds the fresh medium of 600IU/mlIL-2,20ng/mlIL-15 to final volume 1000ml by cell counting.
Within (7) the 11st days, splitting is cultivated, after cell counting, divided in two new culture bag (bag b and bag d) in 1.4:1 ratio respectively by cell suspension in former culture bag (bag a and bag c), four cell culture bags add the fresh medium of 600IU/mlIL-2,20ng/mlIL-15 respectively to final volume 1000ml.
(8) the 15th, 17,19,21 days, cell counting, detected cytoactive, harvested cell (bag a, bag b, bag c, bag d).After the cell gathered in the crops hatches 30 minutes at normal temperatures with anti-EGFR × AntiCD3 McAb bifunctional antibody respectively, for subsequent use with brine cell.
Note: above cell is all at 37 DEG C of 5%CO
2cultivate in incubator.
Embodiment 2
1. monocyte (PBMC) is separated
A () gets 500ul peripheral blood cellanalyzer counting, Activity determination: mixed with physiological saline equal-volume by peripheral blood, add 40mlFicoll lymphocyte separation medium centrifugation monocyte afterwards; Parameter of noncentricity: 1600rpm, 20 degrees Celsius, 20 minutes;
B () gets tunica albuginea, add physiological saline to cumulative volume 80ml, centrifuge washing twice; Parameter of noncentricity: 2000rpm, 20 degrees Celsius, 8 minutes;
C () cell serum-free basic medium re-suspended cell precipitation, utilizes cell counter to carry out cell counting, calculates cell yield.
2.CIK inducing culture
(1) after the PBMC counting be separated, by 9 × 10
5individual/ml density is inoculated in T175 culturing bottle, and adding final concentration is 500ng/mlCD3mAb, 1500ng/mlCD28mAb, 4400IU/mlIFN-γ, 4400IU/mlIL-2,150ng/mlIL-15, supplements serum free medium to 50ml.
(2) the 3rd days, cell counting, added the fresh medium 50ml of 800IU/mlIL-2,25ng/mlIL-15.
(3) the 4th days, cell counting, added the fresh medium 50ml of 800IU/mlIL-2,25ng/mlIL-15.
Within (4) the 6th days, sub-bottle is cultivated, cell counting, after being mixed by cell suspension in former culturing bottle, 1.4:1 is transferred in two new T175 culturing bottles respectively by volume, and be labeled as a.b and c.d respectively, two bottles of fresh mediums adding 800IU/mlIL-2,25ng/mlIL-15 are respectively to 240ml;
Pack in (5) the 8th days is cultivated, cell counting, cell suspension in a.b culturing bottle is loaded 1.8L cell culture bags (bag a) in, add the fresh medium of 800IU/mlIL-2,25ng/mlIL-15 to final volume 1000ml.
Pack in (6) the 9th days is cultivated, and cell suspension in c.d culturing bottle loads in 1.8L cell culture bags (bag c), adds the fresh medium of 800IU/mlIL-2,25ng/mlIL-15 to final volume 1000ml by cell counting.
Within (7) the 11st days, splitting is cultivated, after cell counting, detect cytoactive, divided in two new culture bag (bag b and bag d) in 1.4:1 ratio respectively by cell suspension in former culture bag (bag a and bag c), four cell culture bags add the fresh medium of 800IU/mlIL-2,25ng/mlIL-15 respectively to final volume 1000ml.
(8) the 15th, 17,19,21 days, cell counting, harvested cell (bag a, bag b, bag c, bag d).After the cell gathered in the crops hatches 40 minutes at normal temperatures with anti-EGFR × AntiCD3 McAb bifunctional antibody respectively, for subsequent use with brine cell.
Note: above cell is all at 37 DEG C of 5%CO
2cultivate in incubator.
Embodiment 3
1. monocyte (PBMC) is separated
A () gets 500ul peripheral blood cellanalyzer counting, Activity determination: mixed with physiological saline equal-volume by peripheral blood, add 40mlFicoll lymphocyte separation medium centrifugation monocyte afterwards; Parameter of noncentricity: 1600rpm, 20 degrees Celsius, 20 minutes;
B () gets tunica albuginea, add physiological saline to cumulative volume 80ml, centrifuge washing twice; Parameter of noncentricity: 2000rpm, 20 degrees Celsius, 8 minutes;
C () cell serum-free basic medium re-suspended cell precipitation, utilizes cell counter to carry out cell counting, calculates cell yield.
2.CIK inducing culture
(1) after the PBMC counting be separated, by 10 × 10
5individual/ml density is inoculated in T175 culturing bottle, and adding final concentration is 600ng/mlCD3mAb, 1600ng/mlCD28mAb, 4800IU/mlIFN-γ, 4800IU/mlIL-2,160ng/mlIL-15, supplements serum free medium to 50ml.
(2) the 3rd days, cell counting, added the fresh medium 50ml of 1000IU/mlIL-2,30ng/mlIL-15.
(3) the 4th days, cell counting, added the fresh medium 50ml of 1000IU/mlIL-2,30ng/mlIL-15.
Within (4) the 6th days, sub-bottle is cultivated, cell counting, after being mixed by cell suspension in former culturing bottle, 1.4:1 is transferred in two new T175 culturing bottles respectively by volume, and be labeled as a.b and c.d respectively, two bottles of fresh mediums adding 1000IU/mlIL-2,30ng/mlIL-15 are respectively to 240ml;
Pack in (5) the 8th days is cultivated, cell counting, cell suspension in a.b culturing bottle is loaded 1.8L cell culture bags (bag a) in, add the fresh medium of 1000IU/mlIL-2,30ng/mlIL-15 to final volume 1000ml.
Pack in (6) the 9th days is cultivated, and cell suspension in c.d culturing bottle loads in 1.8L cell culture bags (bag c), adds the fresh medium of 1000IU/mlIL-2,30ng/mlIL-15 to final volume 1000ml by cell counting.
Within (7) the 11st days, splitting is cultivated, after cell counting, divided in two new culture bag (bag b and bag d) in 1.4:1 ratio respectively by cell suspension in former culture bag (bag a and bag c), four cell culture bags add the fresh medium of 1000IU/mlIL-2,30ng/mlIL-15 respectively to final volume 1000ml.
(8) the 15th, 17,19,21 days, cell counting, detected cytoactive, harvested cell (bag a, bag b, bag c, bag d).After the cell gathered in the crops hatches 35 minutes at normal temperatures with anti-EGFR × AntiCD3 McAb bifunctional antibody respectively, for subsequent use with brine cell.
Note: above cell is all at 37 DEG C of 5%CO
2cultivate in incubator.
Embodiment 4
Embodiment 1-3 cultivates gained CIK cell to the Cytotoxicity in vitro rate of EGFR positive tumor cell
(1) by anti-EGFR × AntiCD3 McAb bifunctional antibody (60ng/10
6cells) treatment group and untreated fish group ControlCIK and target cell, add 96 well culture plates in 5 ︰ 1,10 ︰ 1,20 ︰ 1 ratios, simultaneously laying effect groups of cells, target cell group and blank group, and often group establishes 3 parallel holes, is placed in 37 DEG C, 5%CO
2, incubator is cultivated.
Note: anti-EGFR × AntiCD3 McAb bifunctional antibody prepares gained by EGFR antibody and CD3 antibody by chemical coupling method.
(2), after 20h, 100 μ l supernatants are taken out in every hole, then add 10 μ lCCK8, continue to hatch 4-6h.
(3) on enzyme-linked immunosorbent assay instrument, select 450nm wavelength to survey each hole OD value.
(4) parallel hole mean OD value calculation result:
Killing activity (%)=1-(experimental group OD value one effect group OD value/target cell group OD value) × 100%.As shown in Figure 2, the CIK after anti-EGFR × AntiCD3 McAb bifunctional antibody is hatched to the lethal effect of Hela tumour cell comparatively untreated fish group significantly strengthen, when effect target ratio is only 5 ︰ 1, can 60.67% be reached to the kill rate of Hela tumour cell; When imitating target than during for 10:1, can up to 91.26% to the kill rate of Hela tumour cell.
Note: above cell is all at 37 DEG C of 5%CO
2cultivate in incubator.
Table 1 embodiment 1-3 cultivates gained CIK cell number and Cell viability table, and from then on form can find out that the cell count amplification times that embodiment 1 ~ 3 is gathered in the crops reaches more than 190 times, and the motility rate of cell is all greater than 98%, especially best with the result of embodiment 3.Illustrate, the cultural method adopting the embodiment of the present invention to provide carries out the propagation that CIK cell inducing culture effectively can promote CIK cell.
Table 1 embodiment 1-3 cultivates gained CIK cell number and Cell viability table
The foregoing is only preferred embodiment of the present invention, all equalizations done according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
Claims (2)
1. the CIK cell and improve the cultural method that its specificity kills knurl of increasing, it is characterized in that: described method comprises separation and obtains mononuclearcell, gone in culturing bottle, add CD3mAb, CD28mAb, IFN-γ, IL-2, IL-15, within 4th day, add IL-2 and IL-15, within 6th day, sub-bottle is cultivated, 8th, within 9 days, respectively the cell in bottle is gone in 1.8L cell culture bags, add IL-2 and IL-15, IL-2 and IL-15 was added in the cultivation of cell splitting in 11st day, continue induced amplification, harvested cell in 15-21 days; After hatching 30-40 minute under the cell bi-specific antibody normal temperature of results, with collecting cell after brine, namely make the CIK cell preparation that specificity kills knurl.
2. a kind of CIK cell and improve the cultural method that its specificity kills knurl of increasing according to claim 1, is characterized in that: described method specifically comprises as follows:
(1) monocyte is separated:
A () gets 500ul peripheral blood cellanalyzer counting, Activity determination: mixed with physiological saline equal-volume by peripheral blood, add 40mlFicoll lymphocyte separation medium centrifugation monocyte afterwards; Parameter of noncentricity: 1600rpm, 20 degrees Celsius, 20 minutes;
B () gets tunica albuginea, add physiological saline to cumulative volume 80ml, centrifuge washing twice; Parameter of noncentricity: 2000rpm, 20 degrees Celsius, 8 minutes;
C () cell serum-free basic medium re-suspended cell precipitation, utilizes cell counter to carry out cell counting;
(2) CIK inducing culture:
After a monocyte count that () is separated, by 8-10 × 10
5individual/ml density is inoculated in culturing bottle, adding final concentration is 400-600ng/mlCD3mAb, 1400-1600ng/mlCD28mAb, 4000-4800IU/mlIFN-γ, 4000-4800IU/mlIL-2,140-160ng/mlIL-15, supplements serum free medium to 50ml;
(b) the 3rd day, cell counting, adds the fresh medium 50ml of 600-1000IU/mlIL-2,20-30ng/mlIL-15;
(c) the 4th day, cell counting, adds the fresh medium 50ml of 600-1000IU/mlIL-2,20-30ng/mlIL-15;
D () the 6th day sub-bottle is cultivated, cell counting, after being mixed by cell suspension in former culturing bottle, 1.4:1 is transferred in two new T175 culturing bottles respectively by volume, be labeled as a.b and c.d respectively, two bottles of fresh mediums adding 600-1000IU/mlIL-2,20-30ng/mlIL-15 are respectively to 240ml;
E () pack in the 8th day is cultivated, cell counting, loads cell suspension in a.b culturing bottle in 1.8L cell culture bags-bag a, add the fresh medium of 600-1000IU/mlIL-2,20-30ng/mlIL-15 to final volume 1000ml;
F () pack in the 9th day is cultivated, cell counting, loads cell suspension in c.d culturing bottle in 1.8L cell culture bags-bag c, add the fresh medium of 600-1000IU/mlIL-2,20-30ng/mlIL-15 to final volume 1000ml;
G () the 11st day splitting is cultivated, after cell counting, divided in two new culture bag-bag b and bag d in 1.4:1 ratio respectively by cell suspension in former culture bag-bag a and bag c, four cell culture bags add the fresh medium of 600-1000IU/mlIL-2,20-30ng/mlIL-15 respectively to final volume 1000ml;
(h) the 15th, 17,19,21 days, cell counting, harvest bag a, bag b, bag c, bag d cell, after the cell gathered in the crops hatches 30-40 minute at normal temperatures with anti-EGFR × AntiCD3 McAb bifunctional antibody respectively, for subsequent use with brine cell.
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