CN105087486A - CIK (cytokine-induced killer) cell culture fluid, CIK cell culture method and application of lentinan in CIK cell culture - Google Patents
CIK (cytokine-induced killer) cell culture fluid, CIK cell culture method and application of lentinan in CIK cell culture Download PDFInfo
- Publication number
- CN105087486A CN105087486A CN201510504254.1A CN201510504254A CN105087486A CN 105087486 A CN105087486 A CN 105087486A CN 201510504254 A CN201510504254 A CN 201510504254A CN 105087486 A CN105087486 A CN 105087486A
- Authority
- CN
- China
- Prior art keywords
- cell
- cik
- nutrient solution
- cell culture
- cik cell
- 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
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the biotechnology field, in particular to CIK cell culture fluid, a CIK cell culture method and application of lentinan in CIK cell culture. The CIK cell culture fluid comprises interferon-gamma, CD3 stimulated monoclonal antibodies, interleukin-2, the lentinan and serum-free basal culture media. When the CIK cell culture fluid with the lentinan is used for CIK cell culture, PBMCs (peripheral blood mononuclear cells) can generate lymphocyte activation factors and release helper T cell factors to promote CIK cell multiplication, so that obtained CIK cells have large amplification multiple, high killing activity and high cell surface antigen content. Experiment results show that after 14-day CIK cell culture by the CIK cell culture fluid, the amplification multiple of the CIK cells is larger than 300, in-vitro killing rate (effector-target ratio being 40:1) of the CIK cells is larger than (80+/-2)%, and the number of CIK cell surface antigens (CD3+ and CD56+) is larger than 48%.
Description
Technical field
The present invention relates to biological technical field, particularly relate to cultural method and the application of lentinan in CIK cell is cultivated of a kind of CIK cell nutrient solution and CIK cell.
Background technology
Autoimmune cell treatment is the most ripe tumor biotherapy technology, it isolates mononuclearcell from autologous patient peripheral blood, feed back in patient body after the activation of experiment in vitro room, modification, amplification, play the immunologic function regulating and strengthen patient, and the effect of direct killing tumour cell and virus infected cell.Since being applied to clinical cancer therapy from the eighties in last century, autoimmune cell treatment technology reaches its maturity, and more and more obtains the accreditation of tumour patient and doctor.
Autoimmune cell treatment is different according to mode, mainly can be divided into active immunity treatment and adoptive immunotherapy two kinds.Wherein, active immunity treatment is the peripheral blood gathering patient, separation can activate the factor of patient self anti-tumor in vivo cell, then cultivate in vitro, modify, breed after feed back in patient body, so just can excite rapidly and strengthen the anti-cancer ability of human body self; Adoptive immunotherapy is separated patient's inhibiting tumor cell that this just exists in health, activates in vitro, directly input in patient body antitumor after propagation.
Cytokine-induced killer cell (Cytokine-InducedKiller, CIK) cell is a kind of inhibiting tumor cell for adoptive immunotherapy.CIK cell is owing to expressing CD3+ and CD56+ two kinds of membrane protein molecules simultaneously, therefore the NK cell sample T lymphocyte that is otherwise known as, the non-MHC of the anti-tumor activity powerful with T lymphocyte and NK cell is restricted kills knurl advantage.Therefore, the preferred option that CIK cell is considered to antitumor adoptive cellular immunotherapy of new generation is applied.
At present, common CIK cell in vitro cultural method cultivates peripheral blood mononuclear cell (PBMC) in the serum free medium adding corresponding cytokine, under the stimulation of cytokine, induce PBMC to become CIK cell.But adopt in this way cultivate CIK cell time, the cultivation effect of CIK cell is poor.
Summary of the invention
In view of this, the object of the present invention is to provide cultural method and the application of lentinan in CIK cell is cultivated of a kind of CIK cell nutrient solution and CIK cell, the cultivation effect of CIK cell in nutrient solution provided by the invention is better.
The invention provides a kind of CIK cell nutrient solution, comprise interferon-γ, CD3 excitated type monoclonal antibody, interleukin II, lentinan and serum-free basic medium.
Preferably, the content of described lentinan in nutrient solution is 0.05 ~ 0.3mg/mL.
Preferably, described nutrient solution also comprises serum.
Preferably, the volumn concentration of described serum in nutrient solution is 5 ~ 20%.
Preferably, the content of described interferon-γ in nutrient solution is 200 ~ 1000U/mL; The content of described CD3 excitated type monoclonal antibody in nutrient solution is 10 ~ 80ng/mL; The content of described interleukin II in nutrient solution is 100 ~ 600U/mL.
The invention provides a kind of cultural method of CIK cell, comprise the following steps:
A), use the nutrient solution described in claim 1 ~ 5 any one to cultivate peripheral blood mononuclear cell, obtain CIK cell.
Preferably, described step a) specifically comprises the following steps:
A1), peripheral blood mononuclear cell is resuspended in described nutrient solution, obtains cell suspension;
A2), by described cell suspension inoculation to cell culture container cultivate, obtain CIK cell.
Preferably, the peripheral blood mononuclear cell density in described cell suspension is 0.5 × 10
6~ 1 × 10
6individual/mL.
Preferably, the temperature of described cultivation is 37 DEG C; The CO of described cultivation
2volumetric concentration is 5%; The time of described cultivation is 12 ~ 16 days.
The invention provides the application of a kind of lentinan in CIK cell is cultivated.
Compared with prior art, the invention provides cultural method and the application of lentinan in CIK cell is cultivated of a kind of CIK cell nutrient solution and CIK cell.CIK cell nutrient solution provided by the invention comprises interferon-γ, CD3 excitated type monoclonal antibody, interleukin II, lentinan and serum-free basic medium.The present invention adds lentinan in CIK cell nutrient solution, when using this nutrient solution to carry out CIK cell cultivation, can promote that PBMC produces lymphocyte activating factor (LAF) and release ThF, thus promote the propagation of CIK cell, and then the CIK cell that cultivation is obtained shows large amplification times, strong killing activity and high cell-surface antigens content.Experimental result shows, nutrient solution provided by the invention is adopted to cultivate CIK cell after 14 days, CIK cell amplification times is greater than 300 times, CIK cell in vitro kill rate (effect target is than 40:1) is greater than (80 ± 2) %, and CIK cell surface antigen (CD3+ and CD56+) quantity is greater than 48%.
Embodiment
Be clearly and completely described the technical scheme in the embodiment of the present invention below, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
The invention provides a kind of CIK cell nutrient solution, comprise interferon-γ, CD3 excitated type monoclonal antibody, interleukin II, lentinan and serum-free basic medium.
In the present invention, described interferon-γ (IFN-γ) is a kind of water-soluble dimer cytokine, has antiviral, immunomodulatory and antitumor properties.In an embodiment provided by the invention, described interferon-γ is 200 ~ 1000U/mL at the content of CIK cell nutrient solution; In another embodiment provided by the invention, described interferon-γ is 400 ~ 800U/mL at the content of CIK cell nutrient solution; The source of the present invention to described interferon-γ is not particularly limited, and adopts commercial goods.In the present invention, the Main Function of described interferon-γ is by acting synergistically with other components in nutrient solution, thus promotes the propagation of CIK cell, strengthens the activity of CIK cell killing tumor cells.
In the present invention, described CD3 excitated type monoclonal antibody, also known as CD3 monoclonal antibody, can carry out specific binding with the CD3 molecule of T lymphocytic cell surface.In an embodiment provided by the invention, the content of described CD3 excitated type monoclonal antibody in nutrient solution is 10 ~ 80ng/mL; In another embodiment provided by the invention, the content of described CD3 excitated type monoclonal antibody in nutrient solution is 20 ~ 60ng/mL.The source of the present invention to described CD3 excitated type monoclonal antibody is not particularly limited, and adopts commercial goods.In the present invention, the Main Function of described CD3 excitated type monoclonal antibody is by acting synergistically with other components in nutrient solution, thus promotes the propagation of CIK cell, strengthens the activity of CIK cell killing tumor cells.
In the present invention, described interleukin II (IL-2) is produced by various kinds of cell and acts on a type cytokines of various kinds of cell, owing to being produced by white corpuscle and play a role between white corpuscle at first, so gain the name thus.In an embodiment provided by the invention, the content of described interleukin II in nutrient solution is 100 ~ 600U/mL; In another embodiment provided by the invention, the content of described interleukin II in nutrient solution is 200 ~ 500U/mL.The source of the present invention to described interleukin II is not particularly limited, and adopts commercial goods.In the present invention, the Main Function of described interleukin II is by acting synergistically with other components in nutrient solution, thus promotes the propagation of CIK cell, strengthens the activity of CIK cell killing tumor cells.
In the present invention, described lentinan is the effective active composition extracted from mushroom fruiting body, activeconstituents in lentinan is β-(1-3)-D-dextran with branch, its main chain is made up of the glucosyl group of β-(1-3)-connection, the glucosyl group connected by β-(1-6), in pectination along main chain stochastic distribution.In an embodiment provided by the invention, the content of described lentinan in nutrient solution is 0.05 ~ 0.3mg/mL; In another embodiment provided by the invention, the content of described lentinan in nutrient solution is 0.0625 ~ 0.25mg/mL.The source of the present invention to described lentinan is not particularly limited, and adopts commercial goods.In the present invention, the Main Function of described lentinan is the propagation promoting CIK cell, strengthens the activity of CIK cell killing tumor cells.
In the present invention, described serum-free basic medium is the basic ingredient of CIK cell nutrient solution.In an embodiment provided by the invention, selected serum-free basic medium is the RPMI-1640 substratum that RPMI (RoswellParkMemorialInstitute) provides.In the present invention, the effect of described serum-free basic medium is for cultured cells provides required nutritive ingredient in cell cultivation process.
In an embodiment provided by the invention, described CIK cell nutrient solution also comprises serum.In an embodiment provided by the invention, described serum is autoserum.In an embodiment provided by the invention, the volumn concentration of described serum in nutrient solution is 5 ~ 20%; In another embodiment provided by the invention, the volumn concentration of described serum in nutrient solution is 5 ~ 10%.In the present invention, the Main Function of described serum is for Growth of Cells and propagation provide nutrition in cell cultivation process, promotes the propagation of CIK cell by acting synergistically with other components in nutrient solution simultaneously, strengthens the activity of CIK cell killing tumor cells.
The present invention adds lentinan in CIK cell nutrient solution, when using this nutrient solution to carry out CIK cell cultivation, can promote that PBMC produces lymphocyte activating factor (LAF) and release ThF, thus promote the propagation of CIK cell, and then the CIK cell that cultivation is obtained shows large amplification times, strong killing activity and high cell-surface antigens content.Meanwhile, the present invention, by optimizing the formula of CIK cell nutrient solution, by the synergy of component each in nutrient solution, promotes the propagation of CIK cell further, strengthens the activity of CIK cell killing tumor cells.Experimental result shows, nutrient solution provided by the invention is adopted to cultivate CIK cell after 14 days, CIK cell amplification times is greater than 300 times, CIK cell in vitro kill rate (effect target is than 40:1) is greater than (80 ± 2) %, and CIK cell surface antigen (CD3+ and CD56+) quantity is greater than 48%.
The invention provides a kind of cultural method of CIK cell, comprise the following steps:
A), use the nutrient solution described in technique scheme to cultivate peripheral blood mononuclear cell, obtain CIK cell.
In cultural method provided by the invention, by using described nutrient solution to cultivate peripheral blood mononuclear cell, obtain CIK cell, this process specifically comprises the following steps:
A1), peripheral blood mononuclear cell is resuspended in described nutrient solution, obtains cell suspension;
A2), by described cell suspension inoculation to cell culture container cultivate, obtain CIK cell.
In above-mentioned concrete CIK cell cultural method provided by the invention, first that peripheral blood mononuclear cell is resuspended in described nutrient solution.Wherein, described peripheral blood mononuclear cell (PBMC) preferably obtains by the following method:
Peripheral blood mixes with physiological saline, and the mixed solution obtained is added in ficoll (Ficoll), then carries out centrifugal treating to the mixed system be made up of peripheral blood, physiological saline and Ficoll.Wherein, the volume ratio of described peripheral blood and physiological saline is preferably 1 ~ 2:1 ~ 2.The rotating speed of described centrifugal treating is preferably 2000 ~ 3500rpm; The time of described centrifugal treating is preferably 15 ~ 30min.After centrifugal treating terminates, extract the PBMC slice layer in centrifuge tube, extract the extract physiological saline obtained resuspended, after resuspended, carry out centrifugal treating, the rotating speed of described centrifugal treating is preferably 1500 ~ 2000rpm, and the time of described centrifugal treating is preferably 5 ~ 15min.After centrifugal treating terminates, extract the PBMC in centrifuge tube, extract the extract physiological saline obtained resuspended, centrifugal treating is carried out after resuspended, the rotating speed of described centrifugal treating is preferably 1500 ~ 2000rpm, and the time of described centrifugal treating is preferably 5 ~ 15min, after centrifugal end, remove supernatant liquor, obtain peripheral blood mononuclear cell.
In the present invention, the nutrient solution that resuspended described peripheral blood mononuclear cell adopts is introduced hereinbefore, again just repeats no more.After resuspended, the peripheral blood mononuclear cell density obtaining cell suspension is preferably 0.5 × 10
6~ 1 × 10
6individual/mL.
After obtaining cell suspension, cultivate in described cell suspension inoculation to cell culture container.Wherein, the temperature of described cultivation is 37 DEG C; The CO of described cultivation
2volumetric concentration is 5%; The time of described cultivation is preferably 12 ~ 16 days, is more preferably 14 days.In an embodiment provided by the invention, the detailed process of carrying out in described cell suspension inoculation to cell culture container cultivating is:
First described cell suspension inoculation is cultivated in the first cell culture container.Described first cell culture container is preferably T75flask.Treat that in the first cell culture container, cell density is preferably between 10
6~ 3 × 10
6individual/mL time, in the first cell culture container, add nutrient solution, until cell suspension cumulative volume is 50 ~ 75mL.In the present invention, to make in described first cell culture container cell density between 10
6~ 3 × 10
6incubation time spent by individual/mL is generally 3 ~ 6 days, is preferably 5 days.Afterwards, the cell suspension in the first cell culture container is proceeded in the second cell culture container cultivate.Described second cell culture container is preferably T175flask or culture bag.In the process of cultivating in the second cell culture container, preferably control the density of cell in nutrient solution, in described nutrient solution, the density of cell preferably controls to be 10
6~ 3 × 10
6individual/mL.In the present invention, preferably adopt the mode adding described CIK cell nutrient solution in foster container to control the density of cell in nutrient solution, before fluid infusion, in nutrient solution, the density of cell is preferably not more than 3 × 10
6individual/mL, after fluid infusion, in nutrient solution, the density of cell is preferably 0.5 × 10
6~ 1.5 × 10
6individual/mL.In an embodiment provided by the invention, control in the described CIK cell nutrient solution added in the density process of cell in nutrient solution not containing serum.The time that described cell suspension after the first cell culture container is cultivated is cultivated in the second cell culture container is preferably 9 ~ 10 days, is more preferably 9 days.
After cultivation terminates, the cell in collecting cell culture vessel, the cell of collection is the CIK cell that the present invention cultivates acquisition.
CIK cell cultural method provided by the invention with the addition of lentinan in CIK cell culturing process, lentinan can promote that PBMC produces lymphocyte activating factor (LAF) and release ThF, thus promote the propagation of CIK cell, and then the CIK cell that cultivation is obtained shows large amplification times, strong killing activity and high cell-surface antigens content.Meanwhile, cultural method provided by the invention, by optimizing the formula of the nutrient solution used in CIK cell culturing process, promotes the propagation of CIK cell further, strengthens the activity of CIK cell killing tumor cells.Experimental result shows, method provided by the invention is adopted to cultivate CIK cell after 14 days, CIK cell amplification times is greater than 300 times, CIK cell in vitro kill rate (effect target is than 40:1) is greater than (80 ± 2) %, and CIK cell surface antigen (CD3+ and CD56+) quantity is greater than 48%.
The invention provides the application of a kind of lentinan in CIK cell is cultivated.Provided by the invention being applied in CIK cell culturing process with the addition of lentinan, facilitates the propagation of CIK cell.
For the purpose of clearer, be described in detail below by following examples.
Embodiment 1
1) 40ml peripheral blood is extracted, peripheral blood is transferred in 50ml centrifuge tube, mixes with the normal saline dilution of 1 times of volume, be added to Ficoll (Axis-Shield company of Norway slowly, production code member: 1114547), the centrifugal 30min of 3500rpm;
2), after centrifugal end, the PBMC slice layer in centrifuge tube is extracted, resuspended with physiological saline, the then centrifugal 15min of 2000pm;
3), after centrifugal end, remove supernatant liquor, residuum uses physiological saline resuspended again, then the centrifugal 15min of 2000rpm;
4), after centrifugal end, remove supernatant liquor, obtain about 1.5 × 10
7individual PBMC;
5) use the PBMC of the resuspended above-mentioned acquisition of nutrient solution, obtain cell suspension; Described nutrient solution is made up of IFN-γ (genome company commercially available prod), CD3 excitated type monoclonal antibody (genome company commercially available prod), lentinan (genome company commercially available prod), serum and RPMI-1640 substratum, wherein, IFN-γ content is 1000U/ml, CD3 excitated type monoclonal antibody content is 80ng/ml, IL-2 content is 600U/ml, lentinan content is 0.3mg/ml, and serum content is 20% (percent by volume); In described cell suspension, the density of PBMC is 1 × 10
6individual/ml;
6) by cell suspension inoculation in T75flask, at 37 DEG C, 5% (volumetric concentration) CO
2cultivate under condition;
7) observe upgrowth situation every day, within the 5th day, in T75flask, add nutrient solution, until cell suspension cumulative volume is 50 ~ 75mL in T75flask; Described nutrient solution is made up of IFN-γ, CD3 excitated type monoclonal antibody, lentinan and RPMI-1640 substratum, wherein, IFN-γ content is 1000U/ml, CD3 excitated type monoclonal antibody content is 80ng/ml, IL-2 content is 600U/ml, and lentinan content is 0.25mg/ml;
8) cell suspension in T75flask is proceeded in T175flask, adds step 7) nutrient solution that uses; Ensure after fluid infusion that cell density is 0.5 × 10
6~ 1.5 × 10
6in the scope of individual/ml;
9) step 7 was added every 2 days) nutrient solution that uses, also can timed interval of adding of the growing state determination nutrient solution of visual cell, ensure before fluid infusion that the density of cell is not more than 3 × 10
6/ ml, ensures after fluid infusion that cell density is 0.5 × 10
6~ 1.5 × 10
6individual/ml;
10) cultivation was collected by the 14th day cell.
Embodiment 2
1) 40ml peripheral blood is extracted, peripheral blood is transferred in 50ml centrifuge tube, mixes with the normal saline dilution of 1 times of volume, be added to Ficoll (Axis-Shield company of Norway slowly, production code member: 1114547), the centrifugal 20min of 2800rpm;
2), after centrifugal end, the PBMC slice layer in centrifuge tube is extracted, resuspended with physiological saline, the then centrifugal 10min of 1800pm;
3), after centrifugal end, remove supernatant liquor, residuum uses physiological saline resuspended again, then the centrifugal 10min of 1800rpm;
4), after centrifugal end, remove supernatant liquor, obtain 1.8 × 10
7individual PBMC;
5) use the PBMC of the resuspended above-mentioned acquisition of nutrient solution, obtain cell suspension; Described nutrient solution is made up of IFN-γ (genome company commercially available prod), CD3 excitated type monoclonal antibody (genome company commercially available prod), lentinan (genome company commercially available prod), serum and RPMI-1640 substratum, wherein, IFN-γ content is 500U/ml, CD3 excitated type monoclonal antibody content is 50ng/ml, IL-2 content is 500U/ml, lentinan content is 0.25mg/ml, and serum content is 10% (percent by volume); In described cell suspension, the density of PBMC is 1 × 10
6individual/ml;
6) by cell suspension inoculation in T75flask, at 37 DEG C, 5% (volumetric concentration) CO
2cultivate under condition;
7) observe upgrowth situation every day, within the 5th day, in T75flask, add nutrient solution, until cell suspension cumulative volume is 50 ~ 75mL in T75flask; Described nutrient solution is made up of IFN-γ, CD3 excitated type monoclonal antibody, lentinan and RPMI-1640 substratum, wherein, IFN-γ content is 500U/ml, CD3 excitated type monoclonal antibody content is 50ng/ml, IL-2 content is 500U/ml, and lentinan content is 0.25mg/ml;
8) cell suspension in T75flask is proceeded in T175flask, adds step 7) nutrient solution that uses; Ensure after fluid infusion that cell density is 0.5 × 10
6~ 1.5 × 10
6in the scope of individual/ml;
9) step 7 was added every 2 days) nutrient solution that uses, also can timed interval of adding of the growing state determination nutrient solution of visual cell, ensure before fluid infusion that the density of cell is not more than 3 × 10
6/ ml, ensures after fluid infusion that cell density is 0.5 × 10
6~ 1.5 × 10
6individual/ml;
10) cultivation was collected by the 14th day cell.
Embodiment 3
1) 40ml peripheral blood is extracted, peripheral blood is transferred in 50ml centrifuge tube, mixes with the normal saline dilution of 1 times of volume, be added to Ficoll (Axis-Shield company of Norway slowly, production code member: 1114547), the centrifugal 15min of 2000rpm;
2), after centrifugal end, the PBMC slice layer in centrifuge tube is extracted, resuspended with physiological saline, the then centrifugal 5min of 1500pm;
3), after centrifugal end, remove supernatant liquor, residuum uses physiological saline resuspended again, then the centrifugal 5min of 1500rpm;
4), after centrifugal end, remove supernatant liquor, obtain 1.2 × 10
7individual PBMC;
5) use the PBMC of the resuspended above-mentioned acquisition of nutrient solution, obtain cell suspension; Described nutrient solution is made up of IFN-γ (genome company commercially available prod), CD3 excitated type monoclonal antibody (genome company commercially available prod), lentinan (genome company commercially available prod), serum and RPMI-1640 substratum, wherein, IFN-γ content is 200U/ml, CD3 excitated type monoclonal antibody content is 10ng/ml, IL-2 content is 100U/ml, lentinan content is 0.0625mg/ml, and serum content is 5% (percent by volume); In described cell suspension, the density of PBMC is 1 × 10
6individual/ml;
6) by cell suspension inoculation in T75flask, at 37 DEG C, 5% (volumetric concentration) CO
2cultivate under condition;
7) observe upgrowth situation every day, within the 5th day, in T75flask, add nutrient solution, until cell suspension cumulative volume is 50 ~ 75mL in T75flask; Described nutrient solution is made up of IFN-γ, CD3 excitated type monoclonal antibody, lentinan and RPMI-1640 substratum, wherein, IFN-γ content is 200U/ml, CD3 excitated type monoclonal antibody content is 10ng/ml, IL-2 content is 100U/ml, and lentinan content is 0.0625mg/ml;
8) cell suspension in T75flask is proceeded in T175flask, adds step 7) nutrient solution that uses; Ensure after fluid infusion that cell density is 0.5 × 10
6~ 1.5 × 10
6in the scope of individual/ml;
9) step 7 was added every 2 days) nutrient solution that uses, also can timed interval of adding of the growing state determination nutrient solution of visual cell, ensure before fluid infusion that the density of cell is not more than 3 × 10
6/ ml, ensures after fluid infusion that cell density is 0.5 × 10
6~ 1.5 × 10
6individual/ml;
10) cultivation was collected by the 14th day cell.
Comparative example
1) 40ml peripheral blood is extracted, peripheral blood is transferred in 50ml centrifuge tube, mixes with the normal saline dilution of 1 times of volume, be added to Ficoll (Axis-Shield company of Norway slowly, production code member: 1114547), the centrifugal 20min of 3500rpm;
2), after centrifugal end, the PBMC slice layer in centrifuge tube is extracted, resuspended with physiological saline, the then centrifugal 10min of 1500pm;
3), after centrifugal end, remove supernatant liquor, residuum uses physiological saline resuspended again, then the centrifugal 10min of 1500rpm;
4), after centrifugal end, remove supernatant liquor, obtain 1.5 × 10
7individual PBMC;
5) use the PBMC of the resuspended above-mentioned acquisition of nutrient solution, obtain cell suspension; Described nutrient solution is made up of IFN-γ (genome company commercially available prod), CD3 excitated type monoclonal antibody (genome company commercially available prod) and RPMI-1640 substratum, wherein, IFN-γ content is 500U/ml, CD3 excitated type monoclonal antibody content is 20ng/ml, IL-2 content is 500U/ml; In described cell suspension, the density of PBMC is 1 × 10
6individual/ml;
6) by cell suspension inoculation in T75flask, at 37 DEG C, 5% (volumetric concentration) CO
2cultivate under condition;
7) observe upgrowth situation every day, within the 5th day, in T75flask, add step 5) nutrient solution that uses, until cell suspension cumulative volume is 50 ~ 75mL in T75flask;
8) proceeded in T175flask, added step 5) nutrient solution that uses; Ensure after fluid infusion that cell density is 0.5 × 10
6~ 1.5 × 10
6in the scope of individual/ml;
9) step 5 was added every 2 days) nutrient solution that uses, also can timed interval of adding of the growing state determination nutrient solution of visual cell, ensure before fluid infusion that the density of cell is not more than 3 × 10
6/ ml, ensures after fluid infusion that cell density is 0.5 × 10
6~ 1.5 × 10
6individual/ml;
10) cultivation was collected by the 14th day cell.
Embodiment 4
The measure of merit of CIK cell
Embodiment 1 ~ 3 and comparative example are cultivated the CIK cell obtained and are carried out cell count, Cell viability, kill and wound the detection of vigor and cell-surface antigens;
1) survival rate test before and after cell cryopreservation
After the cell of embodiment 1 ~ 3 group and control group is carried out Trypan Blue, carry out cell counting with cell counting count board, calculate cell count and Cell viability, as following table 1
Table 1 cell count and Cell viability table
Can find out that the cell count of embodiment 1 ~ 3 group is far above comparative example from above table, also just illustrate that the amplification times of embodiment is higher than comparative example, the motility rate of cell is also higher than comparative example, and especially the result of embodiment 2 is best.Illustrate, the cultural method adopting the embodiment of the present invention to provide carries out CIK cell and cultivates the propagation that can promote CIK cell.
2) detection of cell killing vigor
Effect target is than the ratio being effector cell and target cell, and detection method is here lactic dehydrogenase enzyme process mainly, the killing activity experiment undertaken by this method, and simple its detecting step of description is as follows:
A. target cell preparation: the target cell of getting cultivation 24 ~ 48h, washs 3 times, finally with complete RPMI-1640 nutrient solution adjustment cell concn to 1 × 10
5individual/ml, for subsequent use.
B. the preparation of effector cell: collect the required CIK cell detected, wash 3 times, finally with complete RPMI-1640 nutrient solution adjustment cell concn to 1 × l0
7individual/ml.
C. effect-target cell effect: get effector cell and each 0.1ml of target cell (effector cell (E)/target cell (T)=100: 1, individual /) and add in the hole of 40 orifice plates, obtain experimental group, every part of sample establishes 3 multiple holes.Laying effect cell Spontaneous release group, target cell Spontaneous release control group and the maximum release control group of target cell (0.1ml target cell+0.1ml1%NP-40 cell pyrolysis liquid) simultaneously, low-speed centrifugal 1000r/min, after 2min, puts 37 DEG C, 5%CO
22h is hatched in incubator.
D. enzymatic reaction: take out culture, drawing each hole supernatant 0.1ml is added in another cultivation plate hole, put 37 DEG C of pre-temperature 10min, every hole adds freshly prepared serum lactic dehydrogenase (LDH) substrate solution 0.1ml, room temperature lucifuge reaction 10 ~ 15min, every hole adds 1mol/L citric acid stop buffer 30 μ l, to stop enzymatic reaction.
E. result calculates: under 570nm wavelength, read each hole OD value with enzyme connection detector, calculate CIK cell and kill and wound vigor, CIK cell kills and wounds vigor (%)=(spontaneous-K562 is spontaneous for experiment-CIK cell)/(maximum-K562 of K562 is spontaneous) × 100%, wherein, experiment is the OD value of experimental group, CIK cell is spontaneous is the OD value of effector cell's Spontaneous release group, K562 is spontaneous is the OD value of target cell Spontaneous release control group, and K562 is the OD value of the maximum release control group of target cell to the maximum.Result reference is in table 2.
3) cell-surface antigens is detected by flow cytometer, and detected result is with reference to table 2.
The detected result of table 2 cell killing vigor and surface antigen
Can find out no matter the CIK cell that the cultural method of embodiment group obtains is from killing activity by the every detected result in table 2, or cell-surface antigens is quantitatively all better than the cultural method of comparative example, illustrate that the cultural method adopting the embodiment of the present invention to provide carries out CIK cell and cultivates the activity that can strengthen CIK cell killing tumor cells.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. a CIK cell nutrient solution, comprises interferon-γ, CD3 excitated type monoclonal antibody, interleukin II, lentinan and serum-free basic medium.
2. nutrient solution according to claim 1, is characterized in that, the content of described lentinan in nutrient solution is 0.05 ~ 0.3mg/mL.
3. nutrient solution according to claim 1, is characterized in that, also comprises serum.
4. nutrient solution according to claim 3, is characterized in that, the volumn concentration of described serum in nutrient solution is 5 ~ 20%.
5. nutrient solution according to claim 1, is characterized in that, the content of described interferon-γ in nutrient solution is 200 ~ 1000U/mL; The content of described CD3 excitated type monoclonal antibody in nutrient solution is 10 ~ 80ng/mL; The content of described interleukin II in nutrient solution is 100 ~ 600U/mL.
6. a cultural method for CIK cell, comprises the following steps:
A), use the nutrient solution described in claim 1 ~ 5 any one to cultivate peripheral blood mononuclear cell, obtain CIK cell.
7. cultural method according to claim 6, is characterized in that, described step a) specifically comprises the following steps:
A1), peripheral blood mononuclear cell is resuspended in described nutrient solution, obtains cell suspension;
A2), by described cell suspension inoculation to cell culture container cultivate, obtain CIK cell.
8. cultural method according to claim 7, is characterized in that, the peripheral blood mononuclear cell density in described cell suspension is 0.5 × 10
6~ 1 × 10
6individual/mL.
9. cultural method according to claim 7, is characterized in that, the temperature of described cultivation is 37 DEG C; The CO of described cultivation
2volumetric concentration is 5%; The time of described cultivation is 12 ~ 16 days.
10. the application of lentinan in CIK cell is cultivated.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510504254.1A CN105087486A (en) | 2015-08-17 | 2015-08-17 | CIK (cytokine-induced killer) cell culture fluid, CIK cell culture method and application of lentinan in CIK cell culture |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510504254.1A CN105087486A (en) | 2015-08-17 | 2015-08-17 | CIK (cytokine-induced killer) cell culture fluid, CIK cell culture method and application of lentinan in CIK cell culture |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105087486A true CN105087486A (en) | 2015-11-25 |
Family
ID=54568822
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510504254.1A Pending CN105087486A (en) | 2015-08-17 | 2015-08-17 | CIK (cytokine-induced killer) cell culture fluid, CIK cell culture method and application of lentinan in CIK cell culture |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105087486A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106520695A (en) * | 2016-12-02 | 2017-03-22 | 中国计量大学 | Additive containing shen-fu polysaccharide and promoting CIK cell proliferation and differentiation, culture medium, culture method and application |
| CN106754702A (en) * | 2016-12-26 | 2017-05-31 | 华东理工大学 | The method of Cytokine-induced killer cells dynamic suspension culture |
| CN107119013A (en) * | 2017-04-14 | 2017-09-01 | 南华生物医药股份有限公司 | A kind of preparation method of enhanced CIK cell and the application of soluble polysaccharide and LBP-X |
| CN107794242A (en) * | 2017-10-11 | 2018-03-13 | 重庆金时代生物技术有限公司 | A kind of CIK cell culture medium |
| CN109706117A (en) * | 2018-12-30 | 2019-05-03 | 深圳光彩生命工程技术有限公司 | A kind of new CIK cell cultural method |
| CN109706118A (en) * | 2018-12-30 | 2019-05-03 | 深圳光彩生命工程技术有限公司 | A kind of combinations of factors of new culture CIK cell |
| CN110592015A (en) * | 2019-09-27 | 2019-12-20 | 中国科学院西双版纳热带植物园 | Paris polyphylla polysaccharide composition for inducing and enhancing CIK cells and application thereof |
-
2015
- 2015-08-17 CN CN201510504254.1A patent/CN105087486A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106520695A (en) * | 2016-12-02 | 2017-03-22 | 中国计量大学 | Additive containing shen-fu polysaccharide and promoting CIK cell proliferation and differentiation, culture medium, culture method and application |
| CN106754702A (en) * | 2016-12-26 | 2017-05-31 | 华东理工大学 | The method of Cytokine-induced killer cells dynamic suspension culture |
| CN106754702B (en) * | 2016-12-26 | 2020-10-30 | 华东理工大学 | Dynamic suspension culture method for cell factor induced killer cells |
| CN107119013A (en) * | 2017-04-14 | 2017-09-01 | 南华生物医药股份有限公司 | A kind of preparation method of enhanced CIK cell and the application of soluble polysaccharide and LBP-X |
| CN107794242A (en) * | 2017-10-11 | 2018-03-13 | 重庆金时代生物技术有限公司 | A kind of CIK cell culture medium |
| CN109706117A (en) * | 2018-12-30 | 2019-05-03 | 深圳光彩生命工程技术有限公司 | A kind of new CIK cell cultural method |
| CN109706118A (en) * | 2018-12-30 | 2019-05-03 | 深圳光彩生命工程技术有限公司 | A kind of combinations of factors of new culture CIK cell |
| CN110592015A (en) * | 2019-09-27 | 2019-12-20 | 中国科学院西双版纳热带植物园 | Paris polyphylla polysaccharide composition for inducing and enhancing CIK cells and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105087486A (en) | CIK (cytokine-induced killer) cell culture fluid, CIK cell culture method and application of lentinan in CIK cell culture | |
| CN102597223B (en) | Process for production of natural killer cells | |
| CN108220239B (en) | A kind of stimulation induction mononuclearcell amplification is composition and its application of gamma delta T cells | |
| JP5358683B2 (en) | Method of growing natural killer cells | |
| CN105087487B (en) | A kind of method of efficient amplification CIK | |
| CN102268405B (en) | Method for auto NK (Natural Killer) cell in-vitro activation and amplification culture and special culture medium thereof | |
| CN109294985A (en) | A method of culture medium system and NK cell expansion ex vivo for NK cell expansion ex vivo | |
| CN102575231B (en) | Process for production of cytokine-induced killer cells | |
| CN106434556B (en) | A kind of method of external evoked amplification I type NKT cell | |
| CN104321425A (en) | Method for inducing and proliferating natural killer cells derived from peripheral blood mononuclear cells | |
| CN104204194A (en) | Method for producing natural killer cells, natural killer cells produced thereby, and composition for treating cancers and infectious diseases containing the same | |
| CN104928243A (en) | Solid tumor patient autologous NK cell separation, excitation, amplification and activity detection method | |
| CN105349489A (en) | Culture method of CIK cell | |
| CN105101978A (en) | Method for producing NK cell-enhancing blood product | |
| CN101314764A (en) | Method for external amplification natural killer cell | |
| CN103525763A (en) | Efficient culture method of CIK (cytokine induced killer) cells | |
| CN105039255A (en) | Addition agent of NKT cell induction culture and method of induction culture | |
| CN107502590A (en) | A kind of method of human umbilical cord's blood candidate stem cell efficient amplification NK cells | |
| CN103436492A (en) | Method for proliferating and activating lymphocytes through serum-free culture | |
| CN105154401A (en) | Method for large-scale culture of NKT cells | |
| CN111662870A (en) | Application of BCG polysaccharide nucleic acid in CIK cell in-vitro culture and preparation of tumor medicine | |
| CN105176926A (en) | Method for amplifying NK cells through in-vitro cultivation | |
| CN110272871A (en) | A kind of stimulation induction mononuclearcell amplification is composition and its application of gamma delta T cells | |
| CN102533649B (en) | Method for easily and efficiently preparing human gamma/deltaT cells | |
| CN103981144A (en) | Preparation method for autologous-serum antigen-sensitized DC-CIK cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20151125 |
|
| RJ01 | Rejection of invention patent application after publication |