CN102517213B - In vitro culture kit for T-lymphocyte cells - Google Patents
In vitro culture kit for T-lymphocyte cells Download PDFInfo
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Abstract
The invention relates to an in vitro culture kit for T-lymphocyte cells, which fundamentally solves the problems of existing T-lymphocyte cell in vitro culture methods that the used materials and culture time are inconsistent, and the culture periods, quality and quantity of effective cells are different. The technical key points are as follows: the content comprises a coating bottle for inducting the differentiation of the T-lymphocyte cells; a CD3 monoclonal antibody, gamma- interferon and interleukin-1 alpha are pre-coated in the coating bottle; the component of a cell factor A, a cell factor B and a cell factor A for stimulating the division and proliferation of the T-lymphocyte cells is 200000 units of interleukin-2, and the component of the cell factor B is 2 million units of interleukin-2. According to the in vitro culture kit for the T-lymphocyte cells, the composite reagent and materials required for the in vitro culture of the T-lymphocyte cells are combined together in a standard way, all the operation is standardized and modularized, so that not only is the working efficiency improved, but also the cell quality and yield difference caused by the operation of different people is reduced.
Description
Technical field
Turn into cytokine induced kill cell the present invention relates to a kind of mononuclearcell for being used to handle in the samples such as human peripheral, Cord blood, hydrothorax, ascites（Cytokine-Induced Killer, abbreviation CIK）Kit, a kind of particularly in vitro culture kit for T lymphocytes, the kit can apply to clinical cytology biological therapy, basic medical research, clinical application research and biotechnology research can also be applied to, it is particularly possible to the research acted on applied to immune system research and tumor-killing.
Background technology
As tumour is increasingly serious to the threat of human survival health, change with rapid changepl. never-ending changes and improvements also occurs for the Therapeutic mode of reply tumour, various novel drugs, new technology, new method emerge in an endless stream, wherein cell biological treatment is after the 4th class tumor therapeuticing method developed after operation, radiation and chemotherapy, as developing direction important in tumor biotherapy.Cancer is the cell disease of neoplastic cells escape immune surveillance system, and the defect of body's immunity is the key of cancer occurrence and development.The antineoplastic immune of body is mainly that T lymphocytes are mediated.Autoimmune cell is fed back in vivo by cell biological treatment technology again through external evoked, differentiation, amplification, bypasses in-vivo tumour dysimmunity mechanism, cancer cell is resisted, suppressed and killed by the immune response of excitating organism.After LAK, infiltration tumor lympha cell and differentiation antigen（Cluster of Differentiation, abbreviation CD）After the killing cell of 3 monoclonal antibodies activation, CIK cytotoxicity is increasingly subject to pay attention to.
CIK cell is to be reported first by Stanford Univ USA Schmidt Dengs for 1991.They are had found under conditions of simulation human internal environment in vitro, and PBLC by cytokine profiles directional induction and can breed and have the heterogeneous effector cell of high efficiency cell cytotoxic activity for a group.Because the cell mass expresses CD3, CD8 and CD56 membrane protein molecule simultaneously, therefore also known as NK sample T lymphocytes, with the restricted advantage for killing knurl of non-principal histocompatibility complex of the powerful anti-tumor activity of T lymphocytes and NK, can specific accumulation play anti-knurl cytotoxicity in tumor by local.CIK cell is gone back to the nest in spleen and lymph node in vivo, is distributed in liver at most, next to that peripheral blood.Wherein CD3+、CD8+、CD56+It is the main effects cell in CIK cell group, the 1-5% of lymphocyte is only accounted in normal human peripheral blood, and after stimulating culture 10-25 days through a variety of factors in vitro, cell quantity can increase by more than 1000 times before relatively cultivating.
Compared with other adoptive immunotherapy cells, CIK cell has the advantages that propagation is fast, it is strong to kill tumor activity, kill knurl and composes wide, Small side effects, influences slight to normal marrow hemopoiesis.There is lethal effect to the tumor cell line of chemotherapy resistance, and the growth on normal hematopoietic colonies cell does not influence.By taking a small amount of mononuclearcell of patient, a large amount of CIK cells are prepared under specified conditions in vitro, directly played after feeding back in vivo and effectively remove function of tumor, it is especially notable to patient's effect after Post operation or chemicotherapy, the small metastatic lesion of residual can be eliminated, prevents cancer cell from spreading and recurring, improves immunity of organisms, and extension patient survival is realized, it is quick to improve the multiple targets such as patients ' life quality.Therefore, CIK is considered as the preferred cell of antitumor adoptive cellular immunotherapy of new generation.Current tumor biotherapy center applications BMDC（Dendritic Cell, abbreviation DC）Combine the method fed back with CIK cell, for disease treatments such as malignant mela noma, kidney, lung cancer, liver cancer, cancer of the esophagus, stomach cancer, carcinomas of urinary bladder.But current each laboratory cultures CIK method, using material, incubation time is inconsistent, causes the cultivation cycle, quality and quantity of effective cell uneven, the unstable risk factors of effect are brought to clinical practice.
In addition, in " a kind of CIK cell and preparation method thereof and the cell preparation " that Chinese Patent Application No. is 200910045790.4, disclosing a kind of CIK cell and preparation method thereof and cell preparation.The inducible factor of its CIK cell recorded is combined as phytohemagglutin phytolectin, anti-CD49d McAb and AntiCD28 McAb.Phytohemagglutin phytolectin used in the program is a kind of mitogen, small lymphocyte can be activated and be converted into lymphoblast, then division growth, discharge lymphokine, the phagocytic function of macrophage is improved, but due to its more difficult purification, and cost is high, so being only difficult to apply to the large-scale use of clinic in the lab as lymphopoietic reagent is stimulated all the time.In " a kind of CIK cell in vitro cultural method " that Chinese Patent Application No. is 200310109565.5, a kind of CIK cell in vitro cultural method is disclosed.The cultural method equally employs two kinds of mitogen joint active cells of phytohemagglutin phytolectin and CD 3-resisting monoclonal antibody, although the stimulus intensity of cell can be strengthened, but this method needs to complete by bioreactor, not only high cost, in-convenience in use, and it is unfavorable for medical research and clinical practice.
To sum up, existing CIK cell（T lymphocytes）Extracorporeal culturing method need to improve, at the same be also more necessary to design the kit for the specification being consistent therewith ensure cultural method it is timely, accurate, be smoothed out.
The content of the invention
It is an object of the invention to provide a kind of in vitro culture kit for T lymphocytes, the kit can be used for the sorting of T lymphocytes in vitro orientation, induction, differentiation, culture, amplification.
The technical solution adopted in the present invention is：This is used for the in vitro culture kit of T lymphocytes, is characterized in that：Kit Contents include：
Sterile centrifugation tube for containing liquid；
Sterile pipette for inhaling tapping body；
Cell sorting liquid for sorting cell；
For the coating bottle of inducer T lymphocyte cell differentiation, CD3 monoclonal antibodies, gamma interferon, interleukin-1 alpha have been coated with advance in the coating bottle；
For the interleukin 2 for stimulating the cell factor A of T lymphocytic cell divisions propagation, cell factor B, the cell factor A composition are 200,000 units, the composition of the cell factor B is the interleukin 2 of 2,000,000 units；
For the steril cell culture bag as culture cell container；
Serum free medium for providing nutrition for cell growth；
Asepsis injector for inhaling tapping body；
Asepsis injector for liquid of being transferred as funnel；
Two-way Blood culture bottle for examining bacterium；
Cell-dispersing agent for preventing cell aggregation；
The aseptic filtration net rolled into a ball for filtration cell；
Feedback bag for containing cell suspension；
The label of information is fed back for marking.
The preparation method of the coating bottle for inducer T lymphocyte cell differentiation is：CD3 monoclonal antibodies, gamma interferon, interleukin-1 alpha is taken to be added in 40ml phosphate buffers, CD3 monoclonal antibodies, gamma interferon, the concentration ratio of three kinds of factors of interleukin-1 alpha are 10:1:1, the mixed solution that total concentration is 1200ng/ml is made, fully mixes；The mixed solution is added in 600ml blake bottles, 4 DEG C stand at least 24 hours, initial coating bottle is made；Bottle will be initially coated with to freeze under the conditions of -40 DEG C 24 hours；Start the compressor of freeze drier, the temperature in machine chamber is down to -40 DEG C, coating bottle bottleneck is unscrewed, hothouse is put into；Vavuum pump is opened, pressure is reached<15Pa, vacuum freeze-drying 20 hours；Open CO2Intake valve, closes vavuum pump；After closing CO after pressure to 110K2Intake valve, takes out coating bottle, tightens lid, seals mouth, 4 DEG C of preservations；Used within the defined shelf-life.
The concentration of the cell factor A is 0.4 ten thousand units/ml, and cell factor B concentration is 0.2 ten thousand units/ml.
The compound method of the cell sorting liquid is：Take 9% Ficoll solution 720ml to be sufficiently mixed with 33.4% cardiografin 30ml, then 7.5g gelatin is added into the mixed liquor, fully mix；The relative density of solution is adjusted with the cardiografin of high concentration or the Ficoll of dilution to 1.077g/ml；30min in the high pressure steam sterilization that temperature is 121 DEG C is finally putting into, room temperature preservation is standby.
The cell-dispersing agent is 10% human serum albumins.
The kit is used to handle the in-vitro directed sorting of the mononuclearcell in human peripheral, Cord blood, hydrothorax, ascites, induction, differentiation, culture, amplification.
The present invention has the advantage that and good effect is：Due to being used to carry out induction differentiation to T lymphocytes present invention employs special combination of cytokines, these three factors are CD3 monoclonal antibodies, gamma interferon, interleukin-1 alpha respectively；The optium concentration ratio of these three factors is 10:1:1, the total final concentration of 1200ng/ml of these three factors in the medium, experiment show that this combination is the very effective mode of inducer T lymphocyte differentiation.Meanwhile, broken up with the method inducer T lymphocyte of the advance coated cell blake bottle of cell factor, add area and the time of T lymphocytes and tokine exposure, be allowed to be easier to receive cell factor stimulation and be induced to differentiate rapidly.In addition, preparing coating bottle present invention employs lyophilized method, this method is very effective for ensureing the activity of cell factor.If this method is applied to industrialization production, it can both extend the shelf-life of coating bottle, the requirement to traffic condition is reduced again, is a kind of excellent way suitable for actual production.
Gamma interferon is by CD4+Or CD8+The homodimer glycoprotein that cell is produced, can directly or indirectly play antitumor action, the activity of enhancing NK, macrophage and cytotoxic T cell by number of ways.Interleukin 2 consumption can be reduced by adding gamma interferon in induction CIK cell forming process；The order and cytotoxic activity that gamma interferon is added simultaneously are closely related, and cytotoxic activity can be improved by first adding addition interleukin 2 after gamma interferon.Because interleukin 2 receptor quantity increase on PMBC can be promoted by first adding gamma interferon, so as to effectively have activated effector cell.CD 3-resisting monoclonal antibody can promote cell to breed as a kind of mitosis accelerator.Early 1980s researchers find that anti-cd 3 antibodies or anti-φt cell receptor antibody can simulate the static T cell of physiologic ligand Activation In Vitro, are allowed to a large amount of propagation, express interleukin 2 receptor, produce endogenous leukocyte interleukin -2.Anti-CD49d McAb has the effect of anti-tumor metastasis, and the growth that antibody suppresses malignant tumour is probably the effect of Immune-enhancing effect, and antibody makes antineoplastic specificity T cell increasing number, increased activity, while inducing for endogenous cytokine is exaggerated this effect.CD 3-resisting monoclonal antibody not only plays an important role in CIK cell incubation, equally has facilitation on killing sensitiveness of the CIK cell to leukaemia and lymthoma is improved.The main mononuclear macrophage by activating of interleukin 1 is produced.The Anticancer effect in vivo mechanism of interleukin 1 is the quantity and function for strengthening T cell and increase tumor infiltrating lymphocyte.Interleukin 1 can be mediated and interleukin 2 receptor is expressed on PMNC, and CIK cellulotoxic effect can be significantly improved when being used in combination with gamma interferon, CD3 monoclonal antibodies, but interleukin 1 does not work to CIK cell amplification.Interleukin 2 is a kind of cell factor of T lymphocytic emiocytosis（The polypeptide being made up of 133 amino acid, relative molecular mass is about 15 × 103）.It is the core substance of human immunity response, with Immune-enhancing effect, antitumor and anti-infective etc. effect.Clinically have been used for the treatment of the illnesss such as kidney, melanoma, lymthoma, lung cancer, stomach cancer, breast cancer, oophoroma, intestinal cancer, carcinoma of urinary bladder, head and neck neoplasm, leukaemia and cancerous thoracoascites.Compared with radiotherapy, chemotherapy, interleukin 2 is slight to normal cellulotoxic side effect, the pain of tumor patient can be mitigated, improve patients ' life quality.In addition interleukin 2 can be used in tuberculosis, hepatitis, AIDS, venereal disease, additive drug addict and other immuuoeorapromised hosts.CIK cell is a group foreign cell group of the common Fiber differentiation of cytokine profiles, and the effect of cytokine profiles is mutually cooperateed with, and the propagation and cytotoxic activity of monofactor pairing effect cell are without effect or less than the synergy of cytokine profiles.The mechanism of synergy is final common activation resting T cells, improves cell expression interleukin 2 receptor and produces the ability of interleukin 2, starts the t cell activation reaction that autocrine pathway interleukin 2 is relied on.
In addition, the present invention is prepared for a kind of efficient cell sorting liquid, the cell sorting liquid energy enough effectively cracks the red blood cell in blood, red blood cell component is fully precipitated, separated with mononuclearcell to be separated.Moreover, kit of the present invention uses serum free medium, and serum free medium can reach biologic cleanliness requirement, definite ingredients, steady quality is easy to Quality Control, the shortcoming of people's AB serum is overcome, the chance of patient's inadvertent contamination can be reduced, the popularization and application for immunization therapy are significant.Compared with other method, the kit has that easy to operate, cell growth is fast, induced efficiency is high, it is lethal it is strong, with low cost, be easy to the outstanding advantages such as large-scale production, can apply to basic research and the clinical application research of BMDC.T lymphocytes in vitro is oriented combining and having formulated operation instructions for complicated reagent required for sorting, induction, differentiation, culture, amplification and consumptive material specification by the kit, all operationss are carried out to specifications, regularized operation, medelling, not only increase operating efficiency, and to reduce and operate the cell quality brought and volume variance between personnel.The kit prepares simple, cell yield height, active strong, suitable for industrialized production, opens the preparation method of new adoptive cell, while will also accelerate the development of immune medical science.The kit improves anti-infection ability once applied to clinical cytology biological therapy, can increase the immunity of non-tumor patient, improves Quality of Life of Tumor Patients and extends patient vitals, clinic is extremely important.For hospital and laboratory, the success rate of increase cell culture also means the saving of cost.T lymphocytes are handled using kit of the present invention, more than 95% CD3 can be harvested+Cell, wherein CD3+CD8+Cell is up to more than 80%, CD3+CD4+Cell reaches more than 10-20%, CD3+CD56+Cell reaches 16-20%.
Brief description of the drawings
Below in conjunction with accompanying drawing, the invention will be further described：
Fig. 1 is CIK cell real scene shooting figure under 40 times of mirrors that culture 4 days is obtained after human peripheral is handled through kit of the present invention；
Fig. 2 is CIK cell real scene shooting figure under 40 times of mirrors that culture 7 days is obtained after human peripheral is handled through kit of the present invention；
Fig. 3 is CIK cell real scene shooting figure under 100 times of mirrors that culture 7 days is obtained after human peripheral is handled through kit of the present invention；
Fig. 4 is CIK cell real scene shooting figure under 40 times of mirrors that culture 9 days is obtained after human peripheral is handled through kit of the present invention；
Fig. 5 is CIK cell real scene shooting figure under 40 times of mirrors that culture 10 days is obtained after human peripheral is handled through kit of the present invention；
Fig. 6 is CIK cell real scene shooting figure under 400 times of mirrors that culture 10 days is obtained after human peripheral is handled through kit of the present invention；
Fig. 7 is the CIK cell obtained after people's hydrothorax is handled through kit of the present invention, is determined through flow cytometer and feeds back the CD45+That irised out in scatter diagram, figure is CD45+Leucocyte in cell, these cell representative samples, including lymphocyte, macrophage etc.；
Fig. 8 is the CIK cell obtained after people's hydrothorax is handled through kit of the present invention, is determined through flow cytometer and feeds back the CD3+That irised out in scatter diagram, figure is CD3+T lymphocytes in cell, these cell representative samples；
Fig. 9 is the CIK cell obtained after people's hydrothorax is handled through kit of the present invention, is determined through flow cytometer and feeds back the CD3+CD8+According to CD8 antigens by CD3 in scatter diagram, figure+Two parts that cell divide into, top is CD3+CD8+Double positive cells, representative is cytotoxic T cell；Lower section is CD3+CD8-Single sun cell；
Figure 10 is the CIK cell obtained after people's hydrothorax is handled through kit of the present invention, is determined through flow cytometer and feeds back the CD4+CD8+According to CD4 and CD8 antigens by CD3 in scatter diagram, figure+Cell is divided into four parts, and upper right side is CD4+CD8+Double positive cells；Lower right is CD4-CD8+Single sun cell, representative is cytotoxic T cell；Lower left is CD4-CD8-Jack to jack adapter cell；Upper left side is CD4+CD8-Single sun cell, representative is helper T lymphocyte.
Embodiment
With reference to Fig. 1 ~ 10 and the present invention is described in detail.Kit of the present invention is specification, the standardized equipment prepared for T culture in vitro of lymphocytes method, and Kit Contents are described in detail below：
The Kit Contents include：Sterile centrifugation tube for containing liquid, 50 × 50ml/；Sterile pipette for inhaling tapping body, 9 × 10ml/ branch；Cell sorting liquid for sorting cell, 7 × 7ml/ pipes；For the coating bottle of Fiber differentiation cell, 1 bottle；The serum free medium of nutrition, 2 × 1L/ bottles needed for for providing growth for cell；Asepsis injector 7 × 5ml/ for inhaling tapping body；For the asepsis injector for liquid of being transferred as funnel, 7 × 50ml/；Cell factor A for stimulating T lymphocytic cell divisions, i.e. interleukin 2,200,000 units/；For the steril cell culture bag as culture cell container, 1 × 2L/；Cell factor B for stimulating T lymphocytic cell divisions, i.e. interleukin 2,2,000,000 units/；Two-way Blood culture bottle for examining bacterium, 2；Cell-dispersing agent for preventing cell aggregation, 5 × 2ml/ branch；The aseptic filtration net rolled into a ball for filtration cell, 5；Sterile feedback bag for containing cell suspension, 5 × 250ml/；The label of information, 5 are fed back for marking.
The preparation of Kit Contents of the present invention and source are as follows：
1st, it is coated with the preparation of bottle
CD3 monoclonal antibodies, gamma interferon, interleukin-1 alpha is taken to be added in 40ml phosphate buffers, CD3 monoclonal antibodies, gamma interferon, the optium concentration ratio of three kinds of factors of interleukin-1 alpha are 10:1:1, the mixed solution that total concentration is 1200ng/ml is made, fully mixes；The mixed solution is added in 600ml blake bottles, 4 DEG C stand at least 24 hours, initial coating bottle is made；Bottle will be initially coated with to freeze under the conditions of -40 DEG C 24 hours；Start the compressor of freeze drier, the temperature in machine chamber is down to -40 DEG C, coating bottle bottleneck is unscrewed, hothouse is put into；Vavuum pump is opened, pressure is reached<15Pa, vacuum freeze-drying 20 hours；Open CO2Intake valve, closes vavuum pump；After closing CO after pressure to 110K2Intake valve, takes out coating bottle, tightens lid, seals mouth, 4 DEG C of preservations；Used within the defined shelf-life.
2nd, the preparation of cell sorting liquid
Take 9% Ficoll solution 720ml to be sufficiently mixed with 33.4% cardiografin 30ml, then 7.5g gelatin is added into the mixed liquor, fully mix.The relative density of solution is adjusted with the cardiografin of high concentration or the Ficoll of dilution to 1.077g/ml.In 121 DEG C of high pressure steam sterilization 30min, room temperature preservation is standby.
3rd, phosphate buffer is bought in Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge（2000ml/ bags）；CD3 monoclonal antibodies are bought in Japanese transgenosis company（5mg/5ml/ branch）；Interleukin-1 alpha is bought in GIBCO companies of the U.S.（100 μ g/ branch）；Interleukin 2 is bought in Beijing Shuanglu Pharmaceutical Co., Ltd.（200000 units/, 2,000,000 units/）；Gamma interferon is bought in Quangang Medicine Co., Ltd., Shandong Prov.（1000000 units/）；Cell-dispersing agent is human serum albumins, is bought in German Ztel Bei Sen biological products Co., Ltd（10g/50ml/ bottles）；Serum free medium is bought in GIBCO companies of the U.S.（1000ml/ bottles）.
4th, centrifuge tube and pipette are bought in Corning Incorporated.
5th, syringe is bought in Shenyang northern new railway Hua Yi materials Co., Ltd.
The extracorporeal culturing method of T lymphocytes of the present invention is mainly used in the sorting of T lymphocytes in vitro orientation, induction, differentiation, culture, amplification；Its manageable biological sample includes human peripheral, Cord blood, hydrothorax, ascites etc.；It can apply to clinical cytology biological therapy, including kidney, malignant mela noma, leukaemia, breast cancer, the carcinoma of the rectum, stomach cancer, lung cancer, the cancer of the esophagus, cervical carcinoma, oophoroma, Huppert's disease, malignant lymphoma（Non-T cell lymthoma）Deng malignancy disease, basic medical research, clinical application research and biotechnology research can also be applied to, it is particularly possible to the research acted on applied to immune system research and tumor-killing.The specific cultural method completed using content in kit of the present invention is as follows：
Embodiment one：
Now using human peripheral as processing sample, using content sorting induction CIK cell in kit of the present invention, and applied to clinical biochemical treatment.Concrete operation step is as follows：
Step one, blood treatments
1. 50ml anticoagulations are injected in 50ml centrifuge tubes, 1600 ~ 2000 × g centrifuges 10min under the conditions of 4 DEG C.It such as can not at once centrifuge, can be placed on 4 DEG C of preservations, but no more than 4 hours.
2. being moved into upper plasma in new 50ml centrifuge tubes with pipette, patient information has been marked.56 DEG C of inactivation 30min.
3. normal saline dilution haemocyte is mixed to 50ml with pipette.It is diluted blood cell processes above.
4. by the haemocyte diluted with 1：1 ratio is slowly injected on every pipe 7ml cell sorting liquid.It is careful not to break interface.
5. under the conditions of 20 DEG C, 2200 ~ 2500 × g centrifugations 20min.Now cell is from top to bottom divided into four layers in centrifuge tube.First layer：Blood plasma or the equal slurry layer of cell tissue；The second layer：Ring-type milky buffy coat；Third layer：Hyaline cell sorts liquid layer；4th layer：Red blood cell layer.First layer liquid is suctioned out with pipette, is discarded；The second confluent monolayer cells are drawn in new 50ml centrifuge tubes.
6. adding physiological saline into the cell newly collected to 50ml, under the conditions of 4 DEG C, 1600 ~ 2000 × g centrifuges 4 ~ 5min.
7. supernatant discarded.Centrifuge tube ttom of pipe is flicked, precipitation is broken up.
8. repeat step 6 ~ 7 is twice.It is cell sorting process above.
9. take the coated cell factor coating of more than 24 hours bottle, once and discarded with physiological saline or culture medium rinse, add 2 ~ 5ml of autologous patient blood plasma.
10. taking 25ml culture medium re-suspended cells, moved it into after fully being mixed with pipette in coating bottle.
11. using 25ml culture medium rinse centrifuge tubes, rinse liquid is moved into coating bottle again.
12. coating bottle is put into 5%CO2, cultivate in 37 DEG C of incubators.Break up incubation above for cell induction.
Step 2 refinement intracellular cytokines A
1. blood treatment second day, takes out coating bottle, observation of cell state from above-mentioned incubator.
2. with 5ml syringe suck fresh culture mediums 2ml dissolving cell factor A, injection is coated with bottle.
3. coating bottle is put into 5%CO2, cultivate in 37 DEG C of incubators.
Step 3 fluid infusion
1. during above-mentioned culture 2 ~ 3 days, as shown in figure 1, growth sign occurs in cell, fresh culture 100ml is added into coating bottle.
2. coating bottle is put into 5%CO2, cultivate in 37 DEG C of incubators.
Step 4 turns bag
1. during above-mentioned culture 1 ~ 2 day, as shown in Figure 2 and Figure 3, when cell is paved with coating bottom of bottle substantially, progress turns bag.
2. blowing down the attached cell in coating bottle with pipette, cell factor B is dissolved with 5ml syringe suck fresh culture mediums 2ml, is added in coating bottle, while adding 2 ~ 5ml of autologous patient blood plasma.
3. taking culture bag, patient information has been marked.
4. remove culture bag pipe cap, 50ml syringe needles and interior bolt.Culture bag inlet is connected with syringe nozzle.
5. the cell culture fluid being coated with bottle is poured into culture bag；With fresh culture rinse coating bottle twice, rinse liquid is poured into culture bag in the lump.
6. fresh culture is added into culture bag to 1000ml.
7. clamping inlet, syringe is removed, pipe cap is covered.
8. culture bag is put into 5%CO2, cultivate in 37 DEG C of incubators.
Step 5 fluid infusion
1. 3-4 days after turn bag, as shown in Fig. 4, Fig. 5, Fig. 6, cell starts a large amount of agglomerating growths, now can moderately rub culture bag, to break up cell mass, cell individual is preferably in contact with the growth factor in culture medium.
2. remove culture bag pipe cap, 50ml syringe needles and interior bolt.Culture bag inlet is connected with syringe nozzle.Add fresh culture 1000ml.
3. clamp liquid inlet.Syringe is removed, pipe cap is covered.
4. culture bag is put into 5%CO2, cultivate in 37 DEG C of incubators.Complete after cell amplification, bacterium to be checked, it is standby.
Step 6 examines bacterium
1. second day after fluid infusion, carry out inspection bacterium.
2. taking out culture bag, culture 5ml is drawn by sample tap with 5ml syringes and injected in two diphase blood culture flasks, one bottle is sent and carries out Sterility testing with third party, and one bottle is used for self-inspection.Two-way Blood culture bottle is placed in 37 DEG C of constant incubators and cultivated.
3. culture bag is put into 5%CO2, cultivate in 37 DEG C of incubators.
If 4. asepsis growth after diphase blood culture flask culture 3 days, it was demonstrated that the culture in culture bag can be used for adoptive therapy.
Step 7 is fed back
1. observation of cell growing state, it is determined that the time of feedback.
2. preparing 50ml centrifuge tubes 8, patient information has been marked.
3. culture is poured into 50ml centrifuge tubes, remaining culture puts back to incubator and continues to cultivate.
4. under the conditions of 4 DEG C, 1600 ~ 2000 × g centrifuges 4 ~ 5min.Supernatant discarded, flicks centrifuge tube ttom of pipe and breaks up cell.
5. with physiological saline re-suspended cell and focus in 2 centrifuge tubes.
6. under the conditions of 4 DEG C, 1600 ~ 2000 × g centrifuges 4 ~ 5min.Supernatant discarded, flicks centrifuge tube ttom of pipe and breaks up cell.
7. with physiological saline re-suspended cell and focus in 1 centrifuge tube.
8. under the conditions of 4 DEG C, 1600 ~ 2000 × g centrifuges 4 ~ 5min.Supernatant discarded, flicks centrifuge tube ttom of pipe and breaks up cell.
9. use physiological saline re-suspended cell.
10. under the conditions of 4 DEG C, 1600 ~ 2000 × g centrifuges 4 ~ 5min.Supernatant discarded, flicks centrifuge tube ttom of pipe and breaks up cell.
11. use physiological saline re-suspended cell.
12. with 5ml syringes a cell-dispersing agent is drawn to inject in new 50ml centrifuge tubes.Sterile screen cloth is put on centrifuge tube.
13. pipette, extract cell suspension is used, in centrifuge tube of the injection equipped with sterile filters.Big cell mass is filtered off by filter screen.
14. fetching transport bag one, patient information has been marked.The mouth of pipe is cut off, 50ml syringes are connected, cell is poured into and adds physiological saline to 150ml.
15. the mouth of pipe is clamped with haemostatic clamp, heat-sealing machine sealing.
Should note in operation it is following some：
, should be softly slow 1. blood is injected on cell separation liquid, it is sure not to break interface.
Rather gas should be made to circulate by loose bottleneck 2. blake bottle is put into after incubator.
3. daily observation of cell upgrowth situation, it is found that when cell colony occurs in the cell in culture bag, soft culture bag breaks up colony.Prevent from largely assembling because of cell and cause the cell death of core.
4. operating process requirement is completed in the local laminar flow superclean bench of ten thousand grades of Clean Operating Labs, to ensure the safety asepsis of whole process.
5. blake bottle should horizontal positioned as far as possible.
Fetch defeated preceding cell suspension and carry out cell count, as a result show, can harvesting number about 5 × 10 after 50ml peripheral blood is treated by the present method9~5×1011It is individual.The cell of a certain example sample harvest treated by the present method is taken to carry out flow cytomery, as a result as shown in Fig. 7 ~ 10.LCA CD45 in cell mass+Cell account for all feed back cells 68.61%（See Fig. 7）, wherein T lymphocyte surface antigens CD3+The ratio of cell reaches 99%（See Fig. 8）, cytotoxic T cell surface antigen CD3+ CD8+Double positive cells account for 68%（See Fig. 9）, helper T lymphocyte surface antigen CD3+ CD4+Double positive cells account for 32%（See Figure 10）, it can thus be seen that the inventive method culture T lymphocytes are with very high yield and purity.
Embodiment two：
The present invention can be additionally used in the external evoked amplification of T lymphocytes in human cord blood.Concrete operation step is as follows：
Step one, blood treatments
1. 80ml anti-freezing Cord bloods are injected separately into 4 50ml centrifuge tubes, 1600 ~ 2000 × g centrifuges 10min under the conditions of 4 DEG C.It such as can not at once centrifuge, can be placed on 4 DEG C of preservations, but no more than 4 hours.
2. discard upper plasma.
3. physiological saline dilutes 4 pipe haemocytes to 50ml respectively, mixed with pipette.It is diluted blood cell processes above.
4. by the haemocyte diluted with 1：1 ratio is slowly injected on every pipe 7ml cell sorting liquid.It is careful not to break interface.
5. under the conditions of 20 DEG C, 2200 ~ 2500 × g centrifugations 20min.The second confluent monolayer cells are drawn in new 50ml centrifuge tubes.
6. adding physiological saline into the cell newly collected to 50ml, under the conditions of 4 DEG C, 1600 ~ 2000 × g centrifuges 4 ~ 5min.
7. supernatant discarded.Centrifuge tube ttom of pipe is flicked, precipitation is broken up.
8. repeat step 6 ~ 7 is twice.It is cell sorting process above.
9. take the coated cell factor coating of more than 24 hours bottle, once and discarded with physiological saline or culture medium rinse.
10. taking 25ml culture medium re-suspended cells, moved it into after fully being mixed with pipette in coating bottle.
11. using 25ml culture medium rinse centrifuge tubes, rinse liquid is moved into coating bottle again.
12. coating bottle is put into 5%CO2, cultivate in 37 DEG C of incubators.Break up incubation above for cell induction.
Step 2 refinement intracellular cytokines A
1. blood treatment second day, takes out coating bottle, observation of cell state from above-mentioned incubator.
2. with 5ml syringe suck fresh culture mediums 2ml dissolving cell factor A, injection is coated with bottle.
3. coating bottle is put into 5%CO2, cultivate in 37 DEG C of incubators.
Step 3 fluid infusion
1. during above-mentioned culture 2 ~ 3 days, fresh culture 100ml is added into coating bottle.
2. coating bottle is put into 5%CO2, cultivate in 37 DEG C of incubators.
Step 4 turns bag
1. during above-mentioned culture 1 ~ 2 day, when cell is paved with coating bottom of bottle substantially, progress turns bag.
2. blowing down the attached cell in coating bottle with pipette, cell factor B is dissolved with 5ml syringe suck fresh culture mediums 2ml, is added in coating bottle.
3. taking culture bag, patient information has been marked.
Subsequent step be the same as Example one.
Embodiment three：
The present invention can be additionally used in the external evoked amplification of T lymphocytes in people's hydrothorax or ascites.Concrete operation step is as follows：
Step one, is taken under the conditions of hydrothorax or ascites 1000ml, 4 DEG C, and 1600 ~ 2000 × g centrifuges 4 ~ 5min.
Step 2 supernatant discardeds, flick centrifuge tube ttom of pipe, break up precipitation.
Step 3 rinses cell 3 times with physiological saline.
Step 4 takes coating bottle, once and is discarded with physiological saline or culture medium rinse.
Step 5 takes 25ml culture medium re-suspended cells, is moved it into after fully being mixed with pipette in coating bottle.
Step 6 25ml culture medium rinse centrifuge tubes, rinse liquid is moved into coating bottle again.
Blake bottle is put into 5%CO by step 72, cultivate in 37 DEG C of incubators.
Subsequent step is not in addition to autologous patient blood plasma is added, other step be the same as Examples one.
Claims (5)
1. a kind of in vitro culture kit for T lymphocytes, it is characterised in that：Kit Contents include：
Sterile centrifugation tube for containing liquid；
Sterile pipette for inhaling tapping body；
Cell sorting liquid for sorting cell, the compound method of the cell sorting liquid is：Take 9%Ficoll solution 720ml to be sufficiently mixed with 33.4% cardiografin 30ml, then 7.5g gelatin is added into the mixed liquor, fully mix；The relative density of solution is adjusted with the cardiografin of high concentration or the Ficoll of dilution to 1.077g/ml；30min in the high-pressure steam sterilizing pan that temperature is 121 DEG C is finally putting into, room temperature preservation is standby；
For the coating bottle of inducer T lymphocyte cell differentiation, CD3 monoclonal antibodies, gamma interferon, interleukin-1 alpha are coated with the coating bottle in advance, the concentration ratio of three kinds of factors is 10：1：1, the final total concentration of mixed liquor being made is 1200ng/ml；
For the interleukin 2 for stimulating the cell factor A of T lymphocytic cell divisions propagation, cell factor B, the cell factor A composition are 200,000 units, the composition of the cell factor B is the interleukin 2 of 2,000,000 units；
For the steril cell culture bag as culture cell container；
Serum free medium for providing nutrition for cell growth；
Asepsis injector for inhaling tapping body；
Asepsis injector for liquid of being transferred as funnel；
Two-way Blood culture bottle for examining bacterium；
Cell-dispersing agent for preventing cell aggregation；
The aseptic filtration net rolled into a ball for filtration cell；
Feedback bag for containing cell suspension；
The label of information is fed back for marking.
2. the in vitro culture kit according to claim 1 for T lymphocytes, it is characterised in that：The preparation method of the coating bottle for inducer T lymphocyte cell differentiation is：CD3 monoclonal antibodies, gamma interferon, interleukin-1 alpha is taken to be added in 40ml phosphate buffers, CD3 monoclonal antibodies, gamma interferon, the concentration ratio of three kinds of factors of interleukin-1 alpha are 10:1:1, the mixed solution that total concentration is 1200ng/ml is made, fully mixes；The mixed solution is added in 600ml blake bottles, 4 DEG C stand at least 24 hours, initial coating bottle is made；Bottle will be initially coated with to freeze under the conditions of -40 DEG C 24 hours；Start the compressor of freeze drier, the temperature in machine chamber is down to -40 DEG C, coating bottle bottleneck is unscrewed, hothouse is put into；Vavuum pump is opened, pressure is reached<15Pa, vacuum freeze-drying 20 hours；Open CO2Intake valve, closes vavuum pump；After closing CO after pressure to 110KPa2Intake valve, takes out coating bottle, tightens lid, seals mouth, 4 DEG C of preservations；Used within the defined shelf-life.
3. the in vitro culture kit according to claim 1 for T lymphocytes, it is characterised in that：The concentration of the cell factor A is 0.4 ten thousand units/ml, and cell factor B concentration is 0.2 ten thousand units/ml.
4. the in vitro culture kit according to claim 1 for T lymphocytes, it is characterised in that：The cell-dispersing agent is 10% human serum albumins.
5. the in vitro culture kit according to claim 1 for T lymphocytes, it is characterised in that：For handling the in-vitro directed sorting of the mononuclearcell in human peripheral, Cord blood, hydrothorax, ascites, induction, differentiation, culture, amplification.
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| CN103436492B (en) * | 2013-08-02 | 2016-03-02 | 北京赛诺泰生物科技有限公司 | By the method for serum-free culture amplifying activated lymphocyte |
| CN108220234B (en) * | 2016-12-09 | 2021-01-26 | 贵州太瑞生诺生物医药有限公司 | In-vitro amplification method of non-sentinel lymph node-derived anti-tumor T cells |
| CN106834228B (en) * | 2017-01-17 | 2021-03-23 | 上海新长安生物科技有限公司 | Method for in vitro amplification of CD8+ T cells and cell subsets thereof |
| CN108004213B (en) * | 2018-01-30 | 2020-09-22 | 北京汇智驰康生物科技有限公司 | Method and kit for rapid amplification of CIK cells |
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