CN102925410A - Method for preparing CIK cell by using heparin anticoagulant plasma - Google Patents
Method for preparing CIK cell by using heparin anticoagulant plasma Download PDFInfo
- Publication number
- CN102925410A CN102925410A CN2012104643773A CN201210464377A CN102925410A CN 102925410 A CN102925410 A CN 102925410A CN 2012104643773 A CN2012104643773 A CN 2012104643773A CN 201210464377 A CN201210464377 A CN 201210464377A CN 102925410 A CN102925410 A CN 102925410A
- Authority
- CN
- China
- Prior art keywords
- cell
- blood plasma
- cik
- mononuclearcell
- centrifugal
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Abstract
The invention discloses a method for preparing a CIK cell by using heparin anticoagulant plasma. The method uses heparin anticoagulant plasma of a cancerous person to induce and amplify lymphocytes in vitro. The method provided by the invention can expanse the CIK cell greatly. The experiment result shows that the number of CIK cells increases significantly from the seventh day to 21th day, and can be up to 40+/-15.0 times of the start number of cells. The number of immune phenotype CD3+CD56+ cells in the CIK cells accounts for high proportion in the total number of cell, and can be up to 10-60% when the cells are cultured to the seventh days to 21th days. The biological activity of the CIK cells is improved. The experiment result shows that the cell killing activity is more than or equal to 70% when the cells are cultured to the seventh days to 21th days.
Description
Technical field
The invention belongs to biological technical field, the blood plasma that is specifically related to a kind of patient's of application anticoagulant heparin prepares the method for CIK cell.
Background technology
Cytokine induced kill cell (Cytokine induced killer, CIK) be with human peripheral blood single nucleus cell (Peripheral blood mononuclear cell, PBMC) external through cytokine profiles (such as anti-CD3McAb, IL-2, IFN-γ, IL-1 α etc.) a group foreign cell take CD3+CD56+ as principal character of combined induction generation, non-major histocompatibility complex (Major HistocompatibilityComplex with the lymphocytic anti-tumor activity of T and NK cell, MHC) the restricted knurl advantage of killing, the natural killer cell therefore be otherwise known as (Nature Killer, NK) sample T lymphocyte (NK-T cells).As a kind of new and effective immunologically competent cell, multiplication capacity is strong, the knurl spectrum is wide extremely, tumor activity is strong extremely because it has, to the responsive incomparable advantage of some other effector cell that waits of multidrug resistant tumour cell, be considered to the first-selected cell of antitumor adoptive cellular immunotherapy of new generation.
Traditional CIK cell expansion ex vivo cultural method adopts the substratum that adds ox source property serum (adopting foetal calf serum) or people AB type serum to cultivate more more, there is pathophorous hidden danger and has intracellular toxin residual and infect the risk of other exogenous disease, there is certain potential safety hazard, causes at the clinical application adoptive cellular immunotherapy and be subject to great limitation.Realize that the CIK adoptive cellular immunotherapy is in clinical applying, must resolve the safety issue that the CIK cell expansion ex vivo is cultivated, this just must adopt self blood plasma or serum free medium to substitute the training method of traditional ox source property serum or people AB type serum, with the chance of minimizing inadvertent contamination.Because the serum free medium price is comparatively expensive, limited the method at adoptive cellular immunotherapy in clinical applying.Therefore, the invention provides a kind of patient's of employing autologous plasma in the external method for preparing efficiently, safely the CIK cell.The method has overcome the shortcoming of traditional method, and the security of CIK adoptive cellular immunotherapy is protected.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, the method of a kind of blood plasma of the patient's of application anticoagulant heparin at vitro culture and amplification CIK cell is provided, when reducing the cell cultures expense, reducing the possibility that infects exogenous disease, significantly improved the biological activity of CIK cell, the security of CIK adoptive cellular immunotherapy is protected, the CIK adoptive cellular immunotherapy has been played positive effect in clinical applying.
The present invention realizes the object of the invention by following concrete technical scheme:
1, the collection of peripheral blood mononuclear cell
Gather the mononuclearcell blood plasma suspension of patient 50-100ml at blood cell separator, total cellular score is 5-10 * 10
7
2, the separation of anticoagulant heparin blood plasma and preparation
Mononuclearcell blood plasma suspension is transferred in several 50ml Falcon centrifuge tubes, centrifugal 5-10min under the 2000-2500rpm, at this moment, it is significantly two-layer up and down that mononuclearcell blood plasma suspension is divided into boundary, and the upper strata is plasma layer, and lower floor is the mononuclearcell layer.At first draw the blood plasma on upper strata, and the interpolation final concentration is 1.0-2.0 * 10
5The heparin sodium of U/L, the jog mixing, after placing-20 ℃ of refrigerators to place 30-60min, the blood plasma that will contain heparin sodium changes in several 50ml Falcon centrifuge tubes, behind the centrifugal 5-10min of 2000-2500rpm, the sucking-off upper plasma also places 4 ℃ of Refrigerator stores for subsequent use, discards the cotton-shaped condensation settling (being mainly scleroproein and Fibrinogen) at the bottom of the centrifuge tube.
3, the separation of peripheral blood mononuclear cell, cultivation and activation
The mononuclearcell layer of lower floor then adds after approximately the mass percent concentration of its 2 times of volumes is 0.9% normal saline dilution suspension, slowly join in several 50mlFalcon centrifuge tubes that are preinstalled with 15-25ml lymphocyte separation medium (available from Beijing Suo Laibao Science and Technology Ltd.) (cell suspension volume equates with the lymphocyte separation medium volume), centrifuge tube is the centrifugal 20-40min of 2000-2500rpm room temperature after strict trim, centrifugal rear mononuclearcell (the Peripheral blood mononuclear cell that draws interfacial layer with flat mouth 10ml suction pipe, PBMC), slowly join in several Falcon centrifuge tubes that are preinstalled with 20-40ml RPMI-1640 substratum (flying generation that biological chemistry goods (Beijing) company limited available from Sai Mo), the centrifugal 5-10min of 2000-2500rpm room temperature, wash collecting cell 2-4 time; Press 2.0-4.0 * 10 through the mononuclearcell of centrifuge washing
6/ mL is suspended in the RPMI-1640 substratum that contains volume percent 10-15% patient anticoagulate plasma, and the interpolation final concentration is 1.0-1.5 * 10
6The rhIFN-γ of U/L, mixing, mixed solution change the aseptic Falcon culturing bottle of 750ml over to, and each culturing bottle inoculation volume is 60-70ml, and the culturing bottle that fills cell is placed 37 ℃, 5%CO
2, cultivate in the incubator of saturated humidity, adding final concentration after 24 hours is 5.0-10.0 * 10
5The rhIL-2 of U/L, final concentration are 5.0-10.0 * 10
5The rhIL-1a of U/L and final concentration are that the mouse-anti people CD3 monoclonal antibody (CD3McAb) of 100-200ug/L continue to be cultivated, and afterwards, add every 2-4 days and fresh to contain volume percent 10-15% patient blood plasma and final concentration is 5.0-10.0 * 10
5The RPMI-1640 substratum of U/L rhIL-2, adjusting simultaneously cell concn is 1.5-2.5 * 10
6/ ml, be cultured to 7-21 days, can begin harvested cell, at this moment, available several 50ml centrifuge tubes are collected the CIK cell suspension for the results part, in the centrifugal 5-8min of 2000-2500rpm, collecting cell, add stroke-physiological saline solution, the centrifugal 5-8min of 2000-2500rpm washs 2-4 time again, be suspended at last in the 250ml stroke-physiological saline solution, namely make with anticoagulant heparin blood plasma and cultivate the CIK cell suspension obtain, be used for injection and use, remaining CIK cells is added and fresh is contained volume percent 10-15% patient blood plasma and final concentration is 5.0-10.0 * 10
5The RPMI-1640 substratum of U/L rhIL-2, adjusting simultaneously cell concn is 1.5-2.5 * 10
6/ ml continues in external enlarged culturing.
4, the amplification situation of CIK cell
The CIK cell of cultivating by aforesaid method obtains a large amount of amplifications, experiment is presented at 1-4 days of cultivation, culturing cell quantity reduces to some extent, afterwards, cell quantity is non-linear growth, and cell is bred gradually after 5-6 days, can be observed cell volume under the inverted microscope and increases, endochylema is few, and karyon is large, circle; Cell is the growth of colony sample; Be cultured to 7-10 days, the cell quantity showed increased, colony like cell agglomerate is larger, is suspended in the substratum, enters thereafter the rapid growth phase, and to cultivating 7-21 days, the CIK cell quantity can reach 40.0 ± 15.0 times of initial culturing cell number.
5, the values of immunophenotyping of CIK cell
Respectively the CIK cell of cultivating different number of days is carried out values of immunophenotyping, the immunophenotype of mensuration comprises: CD45, CD3, CD25, CD15, CD4, CD8, CD16, CD56, CD29, CD28.CIK is the foreign cell group of a group take CD3+CD56+ as principal character, with the restricted tumor activity that kills of the non-MHC of the lymphocytic anti-tumor activity of T and NK cell, the ratio that the CD3+CD56+ cell accounts for total cell count progressively raises with incubation time, be cultured to 7-21 days, can reaching 10-60% and not wait; And, cultivate the CIK cell that obtains with patient's anticoagulant heparin blood plasma, the ratio that the CD3+CD56+ cell accounts for total cell count will be higher than the CIK cell of cultivating with foetal calf serum.
6, CIK activity of cell biology effect detection
Adopting mtt assay to measure external CIK is the cell killing experiment (effect: target=10: 1) of K562 to NK cell Sensitive Tumor Cells, experimental result shows: the CIK cell that adopts anticoagulate plasma to cultivate, cultivating 7-21 days, its cell killing activity all 〉=70% and is higher than CIK cell with foetal calf serum and serum free medium cultivation with the CIK cell killing activity that the nutrient solution that contains anticoagulate plasma obtains.
The present invention is as follows with respect to advantage and the technique effect of prior art:
1) the inventive method had higher amplification times, and experimental data show to use anticoagulant heparin blood plasma to cultivate lymphocyte, and CIK cell quantity showed increased can reach 40.0 ± 15.0 times of initial culturing cell number at 7-21 days;
2) to account for the ratio of total cell count high for immunophenotype CD3+CD56+ cell in the CIK cell that obtains by the inventive method, is being cultured to 7-21 days, can reach 10-60%;
3) the CIK cell of cultivating by the inventive method, biologic activity improves, and experimental result is presented at cultivated 7-21 days, and its cell killing activity is all 〉=70%.
4) the CIK cell for preparing by the inventive method is safe and reliable, can be within external certain time limit continuous enlarged culturing, and activity stabilized, be applicable to batch production.
Description of drawings
Fig. 1 is that the CIK cell that patient's of the present invention anticoagulant heparin blood plasma is cultivated acquisition at amplification in vitro is examined under a microscope schematic diagram, and wherein A is 100 times of enlarged views, and B is 400 times of enlarged views;
Fig. 2 is the immunophenotype result schematic diagram of flow cytometer detection CIK cell of the present invention; Wherein I is the cell scatter diagram; II is the one-parameter figure of CD3; III is the two-parameter figure of CD3 and CD56;
Fig. 3 is that to adopt mtt assay to measure respectively the CIK cell of cultivating the 7th, 14 and the 21st days be the cell killing experimental result schematic diagram of K562 to NK cell Sensitive Tumor Cells;
Fig. 4 is the immunophenotype result schematic diagram of cultivating the CIK cell that same patient obtains with the nutrient solution amplification in vitro that contains different blood plasma, and wherein S1 is the patient among the embodiment 1; S2 is the patient among the embodiment 3; A cultivates the CIK cell that obtains with the nutrient solution amplification in vitro that contains foetal calf serum; B: cultivate the CIK cell that obtains with the nutrient solution amplification in vitro that contains anticoagulant heparin blood plasma;
Fig. 5 is the CIK cell that the inventive method prepares patients with gastric cancer, the contrast schematic diagram of diseased region morphology and this position histopathologic slide under the gastroscope before and after the CIK cell feeds back, wherein A-1 is this patients with gastric cancer morphological feature of diseased region under the gastroscope before the CIK cell therapy; A-2 is the result of histopathologic slide of this patients with gastric cancer diseased region before the CIK cell therapy; B-1 is the morphological feature of this patients with gastric cancer same diseased region under gastroscope behind the CIK cell therapy; B-2 is the result of histopathologic slide of this patients with gastric cancer this diseased region behind the CIK cell therapy.
Specific embodiments
Below by drawings and Examples the present invention is described in further detail, but protection scope of the present invention is not limited to described content, method all adopts ordinary method if no special instructions among the embodiment.
Embodiment 1: postoperative cardiac carcinoma patient's anticoagulant heparin blood plasma is used for cultivating the method for Autologous lymphocyte, and particular content is as follows:
1, clinical data
2, main raw
Hyclone RPMI-1640 flies generation that biological chemistry goods (Beijing) company limited available from Sai Mo; Injection recombinant human interleukin--2 (Recombinant human interleukin-2, rhIL-2) is available from Beijing Sihuan Biopharmaceutical Co., Ltd.; Recombinant human interleukin--1 α (Recombinant human interleukin-1 alpha, rhIL-1 α) is purchased from U.S. PeproTech company; Injection recombinant human interferon gamma (Recombinat human interferon γ, rhIFN-γ) is available from Shanghai KaiMao biological medicine Co., Ltd; The anti-human T cell of injection CD3 mouse monoclonal antibody (Monoclonal antibodyCD3, CD3McAb) available from Wuhan Biological Products Inst.'s human lymphocyte parting liquid available from Beijing Suo Laibao Science and Technology Ltd.; Tissue Culture Flask, culture plate are Corning company (U.S.) product; Forma CO
2Incubator (U.S.); Nikon inverted microscope (Japan); The cell cultures operation is all carried out between the CIK cell manipulation that reaches Good Manufacturing Practice and Quality Control of Drug (GMP).
3, the preparation of CIK cell
Gather patient's mononuclearcell at blood cell separator with the lymphocyte capture program, obtain to contain to add up to 5.0 * 10
7The cell suspension of mononuclearcell and 50ml patient's blood plasma.50ml patient's mononuclearcell blood plasma suspension is transferred in 2 50mlFalcon centrifuge tubes, the centrifugal 5min of 2000rpm, at this moment, it is significantly two-layer up and down that mononuclearcell blood plasma suspension is divided into boundary, and the upper strata is plasma layer, and lower floor is the mononuclearcell layer.At first draw the altogether blood plasma of 40ml of upper strata, and the interpolation final concentration is 1.0 * 10
5The heparin sodium of U/L, the jog mixing, after placing-20 ℃ of refrigerators to place 30min, the blood plasma that will contain heparin sodium changes in 2 50ml Falcon centrifuge tubes, behind the centrifugal 5min of 2000rpm, the sucking-off upper plasma also places 4 ℃ of Refrigerator stores for subsequent use, discards scleroproein and the cotton-shaped condensation settling of Fibrinogen at the bottom of the centrifuge tube;
The mass percent concentration that adds its 2 times of volumes (20ml) in the mononuclearcell layer of the 10ml of lower floor is 0.9% physiological saline, cell is behind abundant mixing, slowly add in 2 50ml Falcon centrifuge tubes that add in advance the 15ml lymphocyte separation medium (cell suspension volume equates with the lymphocyte separation medium volume), the centrifugal 30min of 2000rpm room temperature, draw the mononuclearcell of interfacial layer with flat mouth 10ml suction pipe, slowly join 2 Falcon centrifuge tubes that add in advance 30ml RPMI-1640 substratum, the centrifugal 5min of 2000rpm room temperature, after washing 2 times, collecting cell; Mononuclearcell through centrifuge washing presses 2.0 * 10
6/ mL quantity is suspended in the RPMI-1640 substratum that contains volume percent 10% blood plasma, and the interpolation final concentration is 1.0 * 10
6The rhIFN-γ of U/L, mixing changes the aseptic Falcon culturing bottle of 750ml over to, and each culturing bottle inoculation volume is 60ml, totally 2 bottles.The culturing bottle that fills cell is placed 37 ℃, 5%CO
2Cultivate in the incubator (Forma Therapeutics Inc, USA).Add mouse-anti people's CD3 monoclonal antibody (CD3McAb) 100 ug/L behind the 24h, final concentration is 5.0 * 10
5The rhIL-2 of U/L and final concentration are 5.0 * 10
5The rhIL-1a of U/L continue to cultivate, and adds in per 2 days afterwards to contain volume percent 10% blood plasma and final concentration is 5.0 * 10
5The RPMI-1640 substratum of U/L rhIL-2 1 time, and the adjustment cell concn is 1.5 * 10
6/ ml.Cultivate the 7th day harvested cell, collect the CIK cell suspension with 5 50ml centrifuge tubes, the centrifugal 5min of 2000rpm, collecting cell; Add stroke-physiological saline solution, the centrifugal 5min of 2000rpm washs 2 times again, last collecting cell, be made into 250ml with stroke-physiological saline solution, namely obtain using anticoagulant heparin blood plasma at the CIK of vitro culture cell suspension, after the quality control detection is qualified, namely can be used for the vein adoptive therapy.
5) the amplification situation of CIK cell
The CIK cell of cultivating obtains a large amount of amplifications, and at 1-4 days that cultivate, culturing cell quantity reduced to some extent.Afterwards, cell quantity is non-linear growth, and cell is bred gradually after the 5th day, can be observed cell volume under the inverted microscope and increases, and endochylema is few, and karyon is large, circle.Cell is the growth of colony sample; Cultivating the 5th day, the cell quantity showed increased, colony like cell agglomerate is larger, is suspended in the substratum, enters thereafter the rapid growth phase, and to cultivating the 7th day, the CIK cell quantity has reached 27.6 times (seeing Fig. 1) of initial culturing cell number.
6) the CIK Immunophenotyping detects
Collected the CIK cell at the 20th day that cultivates, adopting ordinary method to carry out immunophenotype detects, with using the CD3 (Beckman Coulter) of FITC mark, CD56 (BD Biosciences Pharmingen) the lucifuge incubated cell of PE mark behind the PBS balance liquid washed cell, 4 ℃, 30min.The antibody that flush away is unnecessary, with flow cytometer (Beckman Culter, MoFlo) detect positive cell, interpretation of result uses Kaluza v1.1 software to analyze, it is this group cell that the door A that wherein sets among the I is illustrated in the cell of analyzing among B and the C, this group cell accounts for 82.85% of total cell count, II is the one-parameter figure of CD3, wherein door C represents that the cell of CD3+ accounts for the analysis of cells group's of institute 73.07%, III is the two-parameter figure of CD3 and CD56, and the result shows that the cell of CD3+CD56+ accounts for the analysis of cells group's of institute 42.04% (seeing Fig. 2).
Embodiment 2: the anticoagulant heparin blood plasma of left breast cancer postoperative patient is used for cultivating the method for Autologous lymphocyte, and particular content is as follows:
1, clinical data
2, main raw
Hyclone RPMI-1640 flies generation that biological chemistry goods (Beijing) company limited available from Sai Mo; Injection recombinant human interleukin--2 (Recombinant human interleukin-2, rhIL-2) is available from Beijing Sihuan Biopharmaceutical Co., Ltd.; Recombinant human interleukin--1 α Recombinant human interleukin-1 alpha, rhIL-1 α) is purchased from U.S. PeproTech company; Injection recombinant human interferon gamma (Recombinat human interferon γ, rhIFN-γ) is available from Shanghai KaiMao biological medicine Co., Ltd; The anti-human T cell of injection CD3 mouse monoclonal antibody (Monoclonal antibodyCD3, CD3McAb) available from Wuhan Biological Products Inst.'s human lymphocyte parting liquid available from Beijing Suo Laibao Science and Technology Ltd.; Tissue Culture Flask, culture plate are Corning company (U.S.) product; Forma CO
2Incubator (U.S.); Nikon inverted microscope (Japan); The cell cultures operation is all carried out between the CIK cell manipulation that reaches Good Manufacturing Practice and Quality Control of Drug (GMP).
3, the preparation of CIK cell
Gather patient's mononuclearcell at blood cell separator with the lymphocyte capture program, obtain to contain to add up to 7.5 * 10
7The cell suspension of mononuclearcell and 75ml patient's blood plasma.75ml patient's mononuclearcell blood plasma suspension is transferred in 2 50mlFalcon centrifuge tubes, the centrifugal 8min of 2250rpm, at this moment, it is significantly two-layer up and down that mononuclearcell blood plasma suspension is divided into boundary, the upper strata is plasma layer, lower floor is the mononuclearcell layer, at first draw the altogether autologous plasma of 65ml of upper strata, and the interpolation final concentration is 1.50 * 10
5The heparin sodium of U/L, the jog mixing, after placing-20 ℃ of refrigerators to place 45min, the blood plasma that will contain heparin sodium changes in 2 50ml Falcon centrifuge tubes, behind the centrifugal 8min of 2250rpm, the sucking-off upper plasma also places 4 ℃ of Refrigerator stores for subsequent use, discards scleroproein and the cotton-shaped condensation settling of Fibrinogen at the bottom of the centrifuge tube;
The mass percent concentration that adds its 2 times of volumes (20ml) in the mononuclearcell layer of the 10ml of lower floor is 0.9% physiological saline, cell is behind abundant mixing, slowly add 2 50ml Falcon centrifuge tubes (cell suspension volume equates with the lymphocyte separation medium volume) that add in advance the 15ml lymphocyte separation medium, the centrifugal 30min of 2250rpm room temperature, draw the mononuclearcell of interfacial layer with flat mouth 10ml suction pipe, slowly join 2 Falcon centrifuge tubes that add in advance 30ml RPMI-1640 substratum, the centrifugal 7.5min of 2250rpm room temperature, after washing 3 times, PBMC is pressed 3.0 * 10
6/ mL quantity is suspended in the RPMI-1640 substratum that contains volume percent 12.5% patient's blood plasma, and the interpolation final concentration is 1.25 * 10
6The rhIFN-γ of U/L changes the aseptic Falcon culturing bottle of 750ml over to, and each culturing bottle inoculation volume is 65ml, totally 2 bottles, the culturing bottle that fills cell is placed 37 ℃, 5%CO
2Cultivate in the incubator (Forma Therapeutics Inc, USA).Add CD3McAb 150ug/L, rhIL-27.5 * 10 behind the 24h
5U/L and rhIL-1 α 7.5 * 10
5U/L.Interpolation in per 3 days afterwards contains volume percent 12% patient's blood plasma and final concentration is 7.5 * 10
5The fresh RPMI-1640 substratum of the rhIL-2 of U/L 1 time, and the adjustment cell concn is 2.0 * 10
6/ ml cultivates the 14th day harvested cell, collects the CIK cell suspension with 6 50ml centrifuge tubes, the centrifugal 6.5min of 2250rpm, collecting cell; Add stroke-physiological saline solution, the centrifugal 6.5min of 2250rpm washs 3 times again, and last collecting cell is suspended in the 250ml stroke-physiological saline solution, namely obtains cultivating the CIK cell suspension that obtains with anticoagulant heparin blood plasma.
4, the amplification situation of CIK cell
The CIK cell of cultivating obtains a large amount of amplifications, and at 1-4 days that cultivate, culturing cell quantity reduced to some extent.Afterwards, cell quantity is non-linear growth, cell is bred gradually after the 5th day, can be observed cell volume under the inverted microscope and increase, endochylema is few, and karyon is large, circle, cell is the growth of colony sample, thereafter enter the rapid growth phase, to cultivating the 14th day, the CIK cell quantity has reached 42.2 times of initial culturing cell number.
5, the CIK Immunophenotyping detects
Collected the CIK cell at the 14th day that cultivates, adopting ordinary method to carry out immunophenotype detects, with distinguishing the following immunophenotype of packet marking behind the PBS balance liquid washed cell: CD45, CD3, CD25, CD15, CD4, CD8, CD16, CD56, CD29, CD28, the lucifuge incubated cell, 4 ℃, 30min.The antibody that flush away is unnecessary detects positive cell with flow cytometer (Beckman Culter, MoFlo), and interpretation of result uses Kaluza v1.1 software to analyze, and the results are shown in Table 1 and table 2.
The values of immunophenotyping result of table 1:CIK cell
* significance is higher than the 0th day (P＜0.001)
The values of immunophenotyping result of table 2:CIK cell
* significance is higher than the 0th day (P＜0.001)
Embodiment 3: the anticoagulant heparin blood plasma of patients with gastric cancer is used for cultivating the method for Autologous lymphocyte, and particular content is as follows:
1, clinical data
Case 3, man, 50 years old, patients with gastric cancer.
2, main raw
Hyclone RPMI-1640 flies generation that biological chemistry goods (Beijing) company limited available from Sai Mo.Injection recombinant human interleukin--2 (Recombinant human interleukin-2, rhIL-2) is available from Beijing Sihuan Biopharmaceutical Co., Ltd..Recombinant human interleukin--1 α (Recombinant human interleukin-1 alpha, rhIL-1 α) is purchased from U.S. PeproTech company.Injection recombinant human interferon gamma (Recombinat human interferon γ, rhIFN-γ) is available from Shanghai KaiMao biological medicine Co., Ltd.The anti-human T cell of injection CD3 mouse monoclonal antibody (Monoclonal antibodyCD3, CD3McAb) available from Wuhan Biological Products Inst.'s human lymphocyte parting liquid available from Beijing Suo Laibao Science and Technology Ltd..Tissue Culture Flask, culture plate are Corning company (U.S.) product.Forma CO
2Incubator (U.S.).Nikon inverted microscope (Japan).The cell cultures operation is all carried out between the CIK cell manipulation that reaches Good Manufacturing Practice and Quality Control of Drug (GMP).
3, the preparation of CIK cell
Gather patient's mononuclearcell at blood cell separator with the lymphocyte capture program, obtain to contain to add up to 10.0 * 10
7The cell suspension of mononuclearcell and 100ml patient's blood plasma.100ml patient's mononuclearcell blood plasma suspension is transferred in 3 50ml Falcon centrifuge tubes, the centrifugal 10min of 2500rpm, at this moment, it is significantly two-layer up and down that mononuclearcell blood plasma suspension is divided into boundary, the upper strata is plasma layer, lower floor is the mononuclearcell layer, at first draw the altogether blood plasma of 80ml of upper strata, and the interpolation final concentration is 2.0 * 10
5The heparin sodium of U/L, the jog mixing, after placing-20 ℃ of refrigerators to place 60min, the blood plasma that will contain heparin sodium changes in 2 50ml Falcon centrifuge tubes, behind the centrifugal 10min of 2500rpm, the sucking-off upper plasma also places 4 ℃ of Refrigerator stores for subsequent use, discards scleroproein and the cotton-shaped condensation settling of Fibrinogen at the bottom of the centrifuge tube;
The mass percent concentration that adds its 2 times of volumes (40ml) in the mononuclearcell layer of the 20ml of lower floor is 0.9% physiological saline, cell is behind abundant mixing, slowly add 3 50ml Falcon centrifuge tubes (cell suspension volume equates with the lymphocyte separation medium volume) that add in advance the 20ml lymphocyte separation medium, the centrifugal 40min of 2500rpm room temperature, draw the mononuclearcell of interfacial layer with flat mouth 10ml suction pipe, slowly join 2 Falcon centrifuge tubes that add in advance 40mI RPMI-1640 substratum, the centrifugal 10min of 2500rpm room temperature, after washing 4 times, PBMC is pressed 4.0 * 10
6/ mL quantity is suspended in the RPMI-1640 substratum that contains volume percent 15.0% patient's blood plasma, and the interpolation final concentration is 1.50 * 10
6The rhIFN-γ of U/L changes the aseptic Falcon culturing bottle of 750ml over to, and each culturing bottle inoculation volume is 70ml, totally 2 bottles, the culturing bottle that fills cell is placed 37 ℃, 5%CO
2Cultivate in the incubator (Forma Therapeutics Inc, USA).Add CD3McAb 200ug/L, rhIL-210.0 * 10 behind the 24h
5U/L and rhIL-1 α 10.0 * 10
5U/L.Interpolation in per 4 days afterwards contains volume percent 15.0% patient's blood plasma and final concentration is 10.0 * 10
5The fresh RPMI-1640 substratum of the rhIL-2 of U/L 1 time, and the adjustment cell concn is 2.5 * 10
6/ ml cultivates the 21st day harvested cell, collects the CIK cell suspension with 6 50ml centrifuge tubes, the centrifugal 8min of 2500rpm, collecting cell; Add stroke-physiological saline solution, the centrifugal 8min of 2500rpm washs 4 times again, and last collecting cell is suspended in the 250ml stroke-physiological saline solution, namely obtains cultivating the CIK cell suspension that obtains with anticoagulant heparin blood plasma.
4, the amplification situation of CIK cell
The CIK cell of cultivating obtains a large amount of amplifications, and at 1-4 days that cultivate, culturing cell quantity reduced to some extent.Afterwards, cell quantity is non-linear growth, and cell is bred gradually after the 6th day, can be observed cell volume under the inverted microscope and increases, and endochylema is few, and karyon is large, circle.Cell is the growth of colony sample, enters thereafter the rapid growth phase, and to cultivating the 21st day, the CIK cell quantity can reach 50.8 times of initial culturing cell number.
5, CIK activity of cell biology effect detection
The CIK cell that adopts mtt assay to measure respectively this prepared patient of the inventive method cultivates the 7th in vitro culture, 14,21 days CIK cell is the cell killing activity of K562 to NK cell Sensitive Tumor Cells, the RPMI-1640 substratum that contains 10% autologous plasma is adopted respectively in this experiment, the RPMI-1640 substratum of 10% foetal calf serum and serum free medium GT551 are at this patient's of cultured and amplified in vitro CIK cell, the effect target ratio of cell killing experiment is 10: 1, experimental result shows: adopt the CIK cell of cultivating from the body anticoagulate plasma, its cell killing activity is cultivating the 7th, 14, reached respectively 90.1% in 21 days, 86.7%, 92.7%, at three time points that detect, its CIK cell in vitro killing activity all is higher than the CIK cell (seeing Fig. 3) that adopts foetal calf serum and serum free medium to cultivate.
6, cultivate same patient's CIK cell with the nutrient solution amplification in vitro that contains different blood plasma, the detection of its immunophenotype
Cultivate the immunophenotype (seeing Fig. 4) of same patient's CIK cell with the nutrient solution amplification in vitro that contains different blood plasma, wherein S1 is the patient among the embodiment 1; S2 is the patient among the embodiment 3; A cultivates the CIK cell that obtains with the nutrient solution amplification in vitro that contains foetal calf serum; B: cultivate the CIK cell that obtains with the nutrient solution amplification in vitro that contains anticoagulant heparin blood plasma; Among the S1 patient, cultivate in the CIK cell that obtains with the nutrient solution amplification in vitro that contains patient's anticoagulant heparin blood plasma, the cell proportion of CD3+CD56+ is 19.91% (S1-B), and cultivate in the CIK cell that obtains with the nutrient solution amplification in vitro that contains foetal calf serum, the cell proportion of CD3+CD56+ only is 9.01% (S1-A); Among the S2 patient, cultivate in the CIK cell that obtains with the nutrient solution amplification in vitro that contains patient's anticoagulant heparin blood plasma, the cell proportion of CD3+CD56+ is 6.77% (S2-B), and cultivate in the CIK cell that obtains with the nutrient solution amplification in vitro that contains foetal calf serum, the cell proportion of CD3+CD56+ only is 2.89% (S2-A); In the two routine patients of two embodiment, the cell proportion of CD3+CD56+ is used foetal calf serum all being higher than with patient's anticoagulant heparin autologous plasma group;
7, the CIK cell therapy cancer of the stomach that adopts the inventive method to prepare
To cultivate the CIK cell that obtains by the anticoagulant heparin blood plasma that aforesaid method makes, a part is in external continuation enlarged culturing, another part was then on CIK injection cell same day, preparation is applicable to the 250ml CIK cell physiological salt aqueous suspensions of patient's treatment in 1 day usage quantity, through quality control detect qualified after, with disposable transfusion set through the vein fractional injection to the patient.The CIK injection cell is 1 times/day, injects continuously 3 days.During injection, 40-60 drips/minute, rocked gently cell suspension in per 5 minutes in the instillation process, avoid cell settlement to pile up, until drip off.
Before and after the treatment under the gastroscope diseased region morphology relatively with the contrast of corresponding position histopathologic slide, experimental result show this patients with gastric cancer behind the CIK cell therapy than the treatment before: diseased region significantly dwindles under the gastroscope, tumour cell significantly reduces (seeing Fig. 5), A-1 and A-2 show it is this patients with gastric cancer morphological feature of diseased region and result of histopathologic slide under the gastroscope before the CIK cell therapy among Fig. 5, visible significantly pathological tissues and than the tumour cell of the large engrain of multinuclear; B-1 and B-2 show the result of histopathologic slide of the morphological feature and the diseased region that are this patients with gastric cancer same diseased region under gastroscope behind the CIK cell therapy, as seen pathological tissues significantly dwindles, and the cellular form of normal gastric mucosa, do not observe tumour cell.
Claims (1)
1. method for preparing the CIK cell with anticoagulant heparin blood plasma is characterized in that carrying out as follows:
(1) collection of the mononuclearcell blood plasma suspension of peripheral blood
Gather the mononuclearcell blood plasma suspension of patient 50-100ml at blood cell separator, total cellular score is 5-10 * 10
7
(2) separation of anticoagulant heparin blood plasma and preparation
With mononuclearcell blood plasma suspension centrifugal 5-10min under 2000-2500rpm, draw supernatant liquid and namely get blood plasma, lower floor is that the mononuclearcell layer is for subsequent use, adds heparin sodium in blood plasma, the interpolation final concentration of heparin sodium is 1-2 * 10
5U/L in-20 ℃ of lower 30-60min that place, then will contain blood plasma centrifugal 5-10min under 2000-2500rpm of heparin sodium behind the mixing, draw upper plasma and place 4 ℃ to save backup;
(3) separation of peripheral blood mononuclear cell, cultivation and activation
The mass percent concentration that adds 2 times of volume of mononuclearcell layer in lower floor's mononuclearcell layer is 0.9% physiological saline, dilution, diluent is added in the equal-volume lymphocyte separation medium, the centrifugal 20-40min of 2000-2500rpm room temperature, the mononuclearcell of centrifugal rear absorption interfacial layer adds in the RPMI-1640 substratum the centrifugal 5-10min of 2000-2500rpm room temperature, wash 2-4 time, mononuclearcell is pressed 2.0-4.0 * 10 after will washing
6Individual/mL concentration is suspended in the RPMI-1640 substratum that contains volume percent 10-15% patient blood plasma, and the interpolation final concentration is 1.0-1.5 * 10 in suspension
6The rhIFN-γ of U/L, mixing, mixed solution place culturing bottle at 37 ℃, 5%CO
2, behind the cultivation 24h, the adding final concentration is 5.0-10.0 * 10 in the incubator of saturated humidity
5The rhIL-2 of U/L, final concentration are 5.0-10.0 * 10
5The rhIL-1a of U/L and final concentration are the mouse-anti people CD3 monoclonal antibody of 100-200ug/L, at 37 ℃, 5%CO
2, continue to cultivate in the incubator of saturated humidity, afterwards, added every 2-4 days and fresh to contain volume percent 10-15% patient blood plasma and final concentration is 5.0-10.0 * 10
5The RPMI-1640 substratum of U/L rhIL-2, adjusting simultaneously cell concn is 1.5-2.5 * 10
6/ ml cultivates harvested cell after 7-21 days, in the centrifugal 5-8min of 2000-2500rpm, collecting cell adds stroke-physiological saline solution, again in the centrifugal 5-8min washing of 2000-2500rpm 2-4 time, collecting cell also is suspended in the stroke-physiological saline solution, namely obtains the CIK cell suspension.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012104643773A CN102925410A (en) | 2012-11-19 | 2012-11-19 | Method for preparing CIK cell by using heparin anticoagulant plasma |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012104643773A CN102925410A (en) | 2012-11-19 | 2012-11-19 | Method for preparing CIK cell by using heparin anticoagulant plasma |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102925410A true CN102925410A (en) | 2013-02-13 |
Family
ID=47640341
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012104643773A Pending CN102925410A (en) | 2012-11-19 | 2012-11-19 | Method for preparing CIK cell by using heparin anticoagulant plasma |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102925410A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107779435A (en) * | 2016-08-30 | 2018-03-09 | 广州市金航生物科技有限公司 | A kind of co-cultivation supernatant containing autologous CIK cell and its application |
| CN113502266A (en) * | 2021-07-12 | 2021-10-15 | 上海南滨江细胞生物科技有限公司 | Culture method for CIK-containing cells in peripheral blood |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1552848A (en) * | 2003-12-19 | 2004-12-08 | 上海伯瑞生物技术发展有限公司 | CIK cell external culturing method |
| CN101037668A (en) * | 2007-03-01 | 2007-09-19 | 蒋敬庭 | Lymphocyte cultivation liquid and method and application |
| CN102154206A (en) * | 2011-01-31 | 2011-08-17 | 郑骏年 | Preparation method of high-purity, high-multiplication capacity and high-cytotoxin activity CIK (cytokine induced kill) cell |
| CN102321581A (en) * | 2011-09-13 | 2012-01-18 | 深圳市博泰生物医学科技发展有限公司 | Preparation method of ascites tumor cell sensitized DC-CIK |
| CN102676454A (en) * | 2012-05-16 | 2012-09-19 | 北京和泽普瑞生物科技有限公司 | Preparation method for CIK (cytokine induced killer) cell of umbilical cord blood source |
| CN102775494A (en) * | 2012-07-10 | 2012-11-14 | 黄建华 | CD3 resistance/CD133 resistance bi-specific antibody and CIK (cytokine-induced killer) cells loaded by CD3 resistance/CD133 resistance bi-specific antibody |
-
2012
- 2012-11-19 CN CN2012104643773A patent/CN102925410A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1552848A (en) * | 2003-12-19 | 2004-12-08 | 上海伯瑞生物技术发展有限公司 | CIK cell external culturing method |
| CN101037668A (en) * | 2007-03-01 | 2007-09-19 | 蒋敬庭 | Lymphocyte cultivation liquid and method and application |
| CN102154206A (en) * | 2011-01-31 | 2011-08-17 | 郑骏年 | Preparation method of high-purity, high-multiplication capacity and high-cytotoxin activity CIK (cytokine induced kill) cell |
| CN102321581A (en) * | 2011-09-13 | 2012-01-18 | 深圳市博泰生物医学科技发展有限公司 | Preparation method of ascites tumor cell sensitized DC-CIK |
| CN102676454A (en) * | 2012-05-16 | 2012-09-19 | 北京和泽普瑞生物科技有限公司 | Preparation method for CIK (cytokine induced killer) cell of umbilical cord blood source |
| CN102775494A (en) * | 2012-07-10 | 2012-11-14 | 黄建华 | CD3 resistance/CD133 resistance bi-specific antibody and CIK (cytokine-induced killer) cells loaded by CD3 resistance/CD133 resistance bi-specific antibody |
Non-Patent Citations (1)
| Title |
|---|
| 撒亚莲: "恶性实体瘤患者自体CIK细胞的体外扩增及初步临床应用", 《实用癌症杂志》, vol. 21, no. 1, 31 January 2006 (2006-01-31), pages 11 - 14 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107779435A (en) * | 2016-08-30 | 2018-03-09 | 广州市金航生物科技有限公司 | A kind of co-cultivation supernatant containing autologous CIK cell and its application |
| CN113502266A (en) * | 2021-07-12 | 2021-10-15 | 上海南滨江细胞生物科技有限公司 | Culture method for CIK-containing cells in peripheral blood |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102268405B (en) | Method for auto NK (Natural Killer) cell in-vitro activation and amplification culture and special culture medium thereof | |
| CN101519646B (en) | CIK cell, as well as preparation method and cell preparation thereof | |
| CN105087487B (en) | A kind of method of efficient amplification CIK | |
| CN101481677B (en) | Method for maturing dendritic cell by in vitro stimulation | |
| CN105316287A (en) | Method for long-term storage and resuscitation culture of adult peripheral blood mononuclear cell | |
| CN103800898A (en) | Tumor specific killer cell preparation and preparation method thereof | |
| CN104593326A (en) | Method for preparing enhanced DC-CIK cell induced by traditional Chinese medicines and application of enhanced DC-CIK cells induced by traditional Chinese medicines | |
| CN107119013A (en) | A kind of preparation method of enhanced CIK cell and the application of soluble polysaccharide and LBP-X | |
| CN108251365A (en) | Immune cell media system | |
| CN106591231B (en) | BCG polysaccharide nucleic acid for promoting proliferation and differentiation of CIK cells, culture medium, culture method and application | |
| CN103301449A (en) | Preparation method of large-scale culture dendritic cell vaccine and application thereof | |
| CN110564683A (en) | Method for co-culture induced amplification of gamma delta T cells and NK cells | |
| CN102827809B (en) | Preparation method of CIK (Cytokine Induced Killer) cells with high proliferation capacity, high cytotoxic activity and high survival rate, associated CIK cells and application | |
| CN104711224A (en) | In-vitro culture method for increasing human Vdelta2 T cell amplification efficiency and application thereof | |
| CN105176926A (en) | Method for amplifying NK cells through in-vitro cultivation | |
| CN105296421B (en) | The T cell and preparation method of a kind of activation of bispecific antibody and application | |
| CN104480069A (en) | Method of carrying out isolated culture on immune cells by virtue of peripheral blood | |
| CN106754704A (en) | The method of the external evoked amplification of immunocyte | |
| CN103981144A (en) | Preparation method for autologous-serum antigen-sensitized DC-CIK cells | |
| CN105969731B (en) | A method of High Fragmentation activity til cell is largely prepared using pernicious Pleural effusions | |
| CN109153974A (en) | Enhance the composition to abnormal cell lethality and its application | |
| CN103013914A (en) | Method for in-vitro culture of killer T cells | |
| CN105861433A (en) | Method for preparing CIK cell preparation with high-efficiency tumor killing property and prepared CIK cell preparation | |
| CN102925410A (en) | Method for preparing CIK cell by using heparin anticoagulant plasma | |
| CN108642013A (en) | From being detached in Cord blood after CD34 candidate stem cells expand culture, induction prepares Dendritic Cells method to one kind on a large scale |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20130213 |