CN109402055A - A kind of DC-CIK cell culture kit and its cultural method - Google Patents

A kind of DC-CIK cell culture kit and its cultural method Download PDF

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CN109402055A
CN109402055A CN201811340439.3A CN201811340439A CN109402055A CN 109402055 A CN109402055 A CN 109402055A CN 201811340439 A CN201811340439 A CN 201811340439A CN 109402055 A CN109402055 A CN 109402055A
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cell
factor
cik
culture
serum
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马玉国
孙营
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Air China Guangzhou Biological Medicine Science And Technology Co Ltd
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Air China Guangzhou Biological Medicine Science And Technology Co Ltd
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Abstract

The present invention relates to field of biotechnology more particularly to a kind of DC-CIK cell culture kits and its cultural method.The invention discloses a kind of DC-CIK cell culture kits, comprising: DC cell non-serum culture medium and CIK cell serum free medium；DC cell non-serum culture medium includes the first cell culture factor and the second cell culture factor, and CIK cell serum free medium includes: the third cell culture factor, the 4th cell culture factor and nutrients.Effective cell content is more in the DC-CIK cell obtained by the kit culture, and killing activity is high, solves in the DC-CIK cell that the cultivate reagent of existing DC-CIK cell obtains that effective cell content is few, the low technical problem of killing activity.

Description

A kind of DC-CIK cell culture kit and its cultural method
Technical field
The present invention relates to field of biotechnology more particularly to a kind of DC-CIK cell culture kits and its cultural method.
Background technique
Immunotherapy of tumors is applied immunology principle and method, improves the immunogenicity and pairing effect cell of tumour cell The sensibility of killing, excitation and enhancing antitumor immunity of organism response, and application immunocyte and effector molecule are transfused host Interior, collaboration body immune system killing tumour inhibits tumour growth.
CIK (Cytokine Induced Killer) cell, is that human peripheral blood mononuclear cell is simulated people in vitro Vivo environment, with a group foreign cell obtained after cytokine profiles co-incubation proliferation.It, which has, significantly identifies and kills The activity of the various tumour cells of body of hurting sb.'s feelings and virus, wherein CD3+CD56+ double positive cells are main effects in CIK cell group Answer cell, with the powerful anti-tumor activity of T lymphocyte and NK cell non-MHC it is restricted kill tumor advantage, therefore claimed again It is a kind of widely applied immunotherapy of tumors means for the T lymphocyte with NK cytosis.
DC (Dendritic Cells) cell is the strongest professional antigen presenting cell of body function, it can efficiently take the photograph It takes, working process and present antigen, the immune response that starting T cell mediates, to mediate powerful specificity antineoplastic cell Immune response.
After DC cell and CIK cell co-culture, it can promote DC and CIK cell be mature, and the proliferation energy of CIK cell can be improved The quantity of power and tumor-killing function and the bis- positive effector cells of CD3+, CD8+ and CD3+CD56+.DC-CIK cell can not only swash Hair, enhancing tumor patient specificity antineoplastic immunity response, effectively remove internal residual, and induce and be immunized in patient's body Memory, to obtain long-term Antineoplastic effect, it is thus possible to prevent and treat conventional therapy (operation, chemotherapy and radiation) residual tumor disease afterwards Recurrence caused by stove.
Currently, DC-CIK is co-cultured, it is that the DC cell of harvest is added in CIK cultivate reagent, to pass through DC cell It is co-cultured with CIK cell, to obtain DC-CIK cell, but existing DC-CIK is co-cultured in the DC-CIK cell that reagent obtains Effective cell content is few, and killing activity is low, and the quantity of the bis- positive effector cells of CD3+, CD8+ and CD3+CD56+ is low.
Summary of the invention
The present invention provides a kind of DC-CIK cell culture kit and its cultural methods, and it is thin to solve existing DC-CIK Effective cell content is few in the DC-CIK cell that the cultivate reagent of born of the same parents obtains, and killing activity is low, CD3+, CD8+ and CD3+CD56+ The low technical problem of the quantity of double positive effector cells.
Its specific technical solution is as follows:
The present invention provides a kind of DC-CIK cell culture kits, comprising: DC cell non-serum culture medium and CIK cell Serum free medium；
DC cell non-serum culture medium includes the first cell culture factor and the second cell culture factor, and CIK cell is without blood Clear culture medium includes: the third cell culture factor, the 4th cell culture factor and nutrients；
First cell factor includes: interleukin-4 (IL-4) and the cell colony stimulating organism factor (GM-CSF)；
Second cell factor includes: IL-4, GM-CSF and tumor necrosis factor α (TNF-α)；
The third cell factor includes: interferon (IFN-γ)；
4th cell factor includes: CD3pure, interleukin 1 α (IL-1 α) and interleukin 2 (IL-2).
Preferably, the concentration of IL-4 is 16.7ng/ml~33.4ng/ml in first cell factor, more preferably The concentration of 33.4ng/ml, GM-CSF are 60ng/ml~120ng/ml, more preferably 120ng/ml.
Preferably, the concentration of IL-4 is 83.5ng/ml~167ng/m in second cell factor, more preferably The concentration of 167ng/m, GM-CSF be 300ng/ml~600ng/ml, more preferably 600ng/ml, TNF-α concentration be 25ng/ Ml~50ng/ml, more preferably 50ng/ml.
Preferably, the concentration of IFN-γ is 2500IU/ml~5000IU/ml in the third cell factor, more preferably 5000IU/ml。
Preferably, the concentration of CD3pure is 250ng/ml~500ng/ml in the 4th cell factor, more preferably 500ng/ml, IL-1 α concentration be 2.5ng/ml~5ng/ml, the concentration of more preferably 5ng/ml, IL-2 be 500IU/ml~ 1000IU/ml, more preferably 1000IU/ml.
CD3pure is the pure CD3 of GMP rank.
Preferably, the nutrients is fetal calf serum autologous plasma, calf serum or autoserum.
Nutrients is preferably autologous plasma, and in the embodiment of the present invention, autologous plasma is the self blood after heat is inactivated and is centrifuged Slurry.
It should be noted that avoiding the application of fetal calf serum as nutriment using autologous plasma, reduces external source and cause Heat source, sensibiligen pollute probability.Foreign serum is not had to clinically.
The present invention also provides the methods using above-mentioned DC-CIK cell culture kit culture DC-CIK cell, including with Lower step:
Step 1: after being cultivated to serum free medium of the peripheral blood mononuclear cells addition containing the first cell factor, The serum free medium containing the second cell factor is added to be cultivated, DC cell is obtained；
Step 2: the serum free medium that the cell factor containing third is added in Xiang Suoshu peripheral blood mononuclear cells is cultivated Afterwards, nutrients is added and the serum free medium containing the 4th cell factor is cultivated, obtain CIK cell；
Step 3: the DC cell being co-cultured with the CIK cell, obtains DC-CIK cell.
Preferably, before step 1 further include: the extraction of plasma treatment and peripheral blood mononuclear cells:
Plasma treatment specifically: first blood is transferred in centrifuge tube from heparin tube, at room temperature 2000rpm~ 2500rpm is centrifuged 10min~15min, and upper plasma is taken to inactivate 30min in 56 DEG C after centrifugation；
The extraction of peripheral blood mononuclear cells the following steps are included:
Step a: separation monocyte: remaining blood sample after drawing blood plasma is diluted with sodium chloride injection, diluted blood Sample is added in the centrifuge tube of lymphocyte separation medium after mixing and is centrifuged, and 2000rpm~2500rpm centrifugation 20min~ 25min；Step b: the acquisition of peripheral blood mononuclear cells: after step a is centrifuged, 4 layers should be divided into centrifuge tube, from top to bottom Be successively physiological saline plasma layer, mononuclear cell layer, lymphocyte separation medium layer, haemocyte layer, take mononuclear cell layer, twice from Precipitating is mononuclearcell after the heart.
It is centrifuged twice specifically:
It being centrifuged for the first time: the monocyte dilution gathered is centrifuged, 1800rpm is centrifuged 10min, 9 drops 7 are risen, Abandon supernatant.
Second of centrifugation: abandoning supernatant after centrifugation, cell is resuspended with 0.9% sodium chloride injection, by the thin of acquisition Born of the same parents are collected into a centrifuge tube, add 0.9% sodium chloride injection to 50ml, mix well, 1600rpm is centrifuged 5min, rises 9 drops 7, abandon supernatant.Collecting obtained precipitating is peripheral blood mononuclear cells.
It should be noted that DC cell and CIK cell need to be cultivated respectively on the same day in step 1, and incubation time is 7 It~8 days；
Specifically, in step 1, DC cell culture first day: peripheral blood mononuclear cells is resuspended with serum free medium It is placed in culture bottle and cultivates, remove suspension cell, leave attached cell, the free serum culture containing the first cell factor is added Base continues to cultivate；
DC cell culture third day: it is subsequent to add the serum free medium containing the first cell factor for culture to third day Continuous culture；
DC cell culture the 6th day: the serum free medium for containing the second cell factor was added to the 6th day in culture, induced DC Cell maturation continues to cultivate.
Preferably, it after addition nutrients and the serum free medium containing the 4th cell factor are cultivated in step 2, obtains Before the CIK cell, further includes: the serum free medium and the nutrients for containing the 5th cell factor is added；
5th cell factor includes the IL-2, more preferably 1000IU/ that concentration is 1000IU/ml~2000IU/ml ml。
In step 2, CIK cell culture first day: the resuspension of peripheral blood mononuclear cells serum free medium was placed on training It supports in bottle, after cultivating 30min~60min, observes the adherent situation of cell, wherein attached cell is used to cultivate DC cell, suspends thin Born of the same parents cultivate CIK cell, and by the media transfer containing suspension cell into new culture bottle, the nothing of the cell factor containing third is added Blood serum medium is cultivated；
CIK cell culture second day: the autologous plasma after heat is inactivated and is centrifuged being added into culture bottle and containing the 4th cell The serum free medium of the factor is cultivated；
CIK cell culture the 4th day: culture observed that cell mass occurred, cell quantity increases, culture medium face to the 4th day When discoloration is orange, cell is subjected to biography bottle, by 1 T75cm2It is 2 T175cm that bottle, which passes,2Bottle, and be added fresh thin containing the 5th The serum free medium of intracellular cytokine and heat inactivate and be centrifuged after blood plasma, to meet the needs of cell growth.
Wherein, above-mentioned culture is in 37 DEG C, 5%CO2It is carried out in incubator, culture bottle T75cm2Culture bottle, serum-free training The serum free medium that base is GT-T551H3 is supported, in addition to the serum free medium containing the 5th cell factor, other culture mediums are both needed to It just can be used after 0.22 μm of syringe filters filters.
Preferably, step 3 specifically: DC cell is added to CIK by cultivate respectively the 7th day of DC cell and CIK cell In Tissue Culture Flask, culture bottle is washed twice with the serum free medium containing the 5th cell factor, is mixed.
Preferably, step 3 obtains the DC-CIK cell at feedback 1~2 day, also needs that programmed death receptor 1 is added (PD-1, programmed cell death protein 1).
Preferably, the dosage of the PD-1 is 5mg~10mg, more preferably 10mg.
It should be noted that adding the PD-1 factor into cell in the present invention before cell is fed back, reducing tumour cell Escape, can more effective killing tumor cell.
Preferably, after step 3, further includes: amplification cultivation.
Amplification cultivation specifically:
Culture was to the 8th day: observation cell state, cell mass quantity increase, and cell quantity increases, and culture medium color becomes When orange, the cell suspension in Tissue Culture Flask is transferred in cell culture bags by syringe sleeve.Contain the 5th with fresh The serum free medium of cell factor washs culture bottle, and after washing culture medium is incorporated into cell culture bags, autologous plasma is added；
Culture was to the 11st day: culture medium color obviously turns yellow, and cell quantity increased significantly, and adds the nothing that IL-2 has been added Blood serum medium；
Culture was to the 13rd day: PD-1 being added in cell culture bags and is cultivated.
It cultivates to the 15th day collection cell.
It should be noted that regular replenishment contains the serum free medium of the 5th cell factor in the present invention, make present invention side Effective cell content is more in the DC-CIK cell that method culture obtains after 14 days, and killing activity is high.
As can be seen from the above technical solutions, the invention has the following advantages that
The present invention provides a kind of DC-CIK cell culture kit, in the DC-CIK cell which obtains Effective cell content is more, and killing activity is high, by experimental data it is found that CD3+CD56+ cell proportion is 29.8%, CD3+ cell Ratio is that 89.5%, CD3+CD8+ cell proportion is killing rate of 81.8%, the DC-CIK cell to A549 cell and HUH7 cell For 42% and 39%.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this Some embodiments of invention without any creative labor, may be used also for those of ordinary skill in the art To obtain other attached drawings according to these attached drawings.
Fig. 1 is CIK cell Phenotypic examination figure provided by Embodiment 2 of the present invention；
Fig. 2 is killing rate of the DC-CIK cell that provides of the embodiment of the present invention three to A549 cell；
Fig. 3 is killing rate of the DC-CIK cell that provides of the embodiment of the present invention three to HUH7 cell.
Specific embodiment
The embodiment of the invention provides a kind of DC-CIK cell culture kit and its prepare cultural method, for solving Effective cell content is few in the DC-CIK cell that the cultivate reagent of existing DC-CIK cell obtains, and the low technology of killing activity is asked Topic.
In order to make the invention's purpose, features and advantages of the invention more obvious and easy to understand, below in conjunction with the present invention Attached drawing in embodiment, technical scheme in the embodiment of the invention is clearly and completely described, it is clear that disclosed below Embodiment be only a part of the embodiment of the present invention, and not all embodiment.Based on the embodiments of the present invention, this field Those of ordinary skill's all other embodiment obtained without making creative work, belongs to protection of the present invention Range.
The A jacket cell factor used in DC-CIK cultural method provided by the invention is DC-CIK prepared by other researchs Cell culture kit, other raw materials used in preparation method of the present invention and reagent are available on the market.
Just a kind of DC-CIK cell culture kit and its cultural method provided by the present invention are described further below.
The culture of one DC-CIK cell of embodiment
Prepare before operation:
Blower self-cleaning 30min；Centrifugation electromechanical source is opened after into clean area；It opens water-bath and is transferred to 56 DEG C；With 75% Alcohol wipe superclean bench, and open ultraviolet light irradiation 30 minutes；It leaves and opens the ultraviolet lamp of clean area after clean area and carry out Disinfection.
1, whole blood separation and plasma treatment: blood is transferred in centrifuge tube from heparin tube with pipette, is put into centrifugation It in machine, is centrifuged after trim, at room temperature, 2500rpm is centrifuged 10min, and after centrifugation, upper plasma is shifted for 9 reduction of speed 2 of raising speed Into centrifuge tube, lid is covered tightly, membrana oralis is sealed, is placed in 56 DEG C of water-baths and inactivates 30min, every 5min or more in inactivation process It is mixed by inversion blood plasma 3~4 times, can sufficiently be inactivated to blood plasma in centrifuge tube.
2, peripheral blood mononuclear cells is extracted
(1) it separates monocyte: isometric physiological saline being added in obtaining the remaining blood sample after blood plasma, piping and druming is uniform Obtain dilution blood sample；Dilution blood sample is added slowly in the centrifuge tube for filling 20ml lymphocyte separation medium with pipette, it will Blood after dilution is carefully added slowly to lymphocyte separation medium upper layer, makes sure to keep in mind that blood and lymphocyte separation medium cannot be destroyed Interface, in order to avoid influence separating effect.After addition, 2500rpm is centrifuged 20min, 9 reduction of speed 2 of raising speed；
The good sample of above-mentioned centrifugation is gently taken out into centrifuge and is put into Biohazard Safety Equipment, it can be seen that sample is in centrifuge tube Four layers are roughly divided into, is followed successively by physiological saline plasma layer, mononuclear cell layer, lymphocyte separation medium layer, granulocyte from top to bottom Red blood cell layer；It is discarded that physiological saline plasma layer is carefully sucked out with pipette.Then along centrifugation tube wall carefully by remaining physiological saline Plasma layer, mononuclear cell layer and the absorption of lymphocyte separation medium layer are transferred in several centrifuge tubes, are added physiological saline, are obtained monokaryon Cell diluent；
(2) monocyte washs: the monocyte dilution that upper step gathers being centrifuged, 1800rpm centrifugation 10min, rises 9 drops 7, abandons supernatant, and cell is resuspended with 0.9% sodium chloride injection, mixes；
(3) it obtains monocyte: gained cell being collected into a centrifuge tube, adds 0.9% sodium chloride injection extremely 50ml is mixed well, and 1600rpm is centrifuged 5min, is risen 9 drops 7, is abandoned supernatant.Collecting obtained precipitating is the single core of peripheral blood Cell；
3, DC cell culture
(1) Day1: after the peripheral blood mononuclear cells of above-mentioned acquisition is resuspended with 20ml serum free medium GT-T551H3 It is transferred to T75cm230min is cultivated in culture bottle, takes out the suspension cell in bottle to new T75cm after 30min2In culture bottle CIK cell is cultivated, the attached cell in former bottle cultivates DC cell, and 30ml IL-4 containing 33.4ng/ml and 120ng/ml GM- is added The serum free medium of CSF is added from the culture bottle back side, to prevent from blowing off attached cell, then in 5%, 37 DEG C of titanium dioxide It is cultivated in carbon incubator.Culture medium is filtered through 0.22 μm of syringe filters, continues to cultivate；
(2) Day3: culture the 3rd day, the serum-free training of addition 20ml IL-4 containing 33.4ng/ml and 120ng/ml GM-CSF Base GT-T551H3 to be supported, is added from the culture bottle back side, culture medium filters through 0.22 μm of syringe filters, in 37 DEG C, 5%CO2Culture Continue to cultivate in case；
(3) Day6: be added 167ng/ml containing 10ml IL-4,600ng/ml GM-CSF and 50ng/ml TNF-α without blood Clear culture medium GT-T551H3, induces DC cell maturation, continues to cultivate, culture medium is filtered through 0.22 μm of syringe filters；
4, CIK cell culture
(1) Day1: peripheral blood mononuclear cells is resuspended with serum free medium GT-T551H3, obtains cell suspension.By cell Suspension is transferred to T75cm2In culture bottle, after cultivating 30min, the culture medium suction containing suspension cell is transferred to pipette New T75cm2In culture bottle, the serum free medium GT-T551H3 of 20ml IFN-γ containing 5000IU/ml is added, screws ventilative Bottle cap is placed in 37 DEG C, 5%CO2Incubator in carry out cell culture, culture medium is filtered through 0.22 μm of syringe filters；
(2) Day2:4000rpm is centrifuged the blood plasma after the inactivation of 10min step 1, and supernatant is transferred to new centrifuge tube.From Cell is taken out in incubator, and autologous plasma 5ml and CD3pure containing 500ng/ml, 5ng/ml IL-1 α and 1000IU/ml is added The 20ml serum free medium GT-T551H3 of IL-2, continues to cultivate, and culture medium is filtered through 0.22 μm of syringe filters；
(3) Day4: after cell culture three days, observe under the microscope: cell mass occurs, and cell quantity increases, and cultivates When base color becomes orange, cell is transferred to other two T175cm completely2In culture bottle, and fresh addition is added Autologous plasma 7.5ml after the serum free medium of 1000IU/ml IL-2 and inactivation and centrifugation, to meet the need of cell growth It wants；
5, DC cell and CIK cell mixed culture
Day7: with the cell of pipette piping and druming DC Tissue Culture Flask, DC cell is assigned into two CIK cell culture bottles In, the serum free medium GT-T551H3 of the fresh IL-2 containing 1000IU/ml is added, addition volume total amount is 50ml wash bottle two It is secondary to be imported into two CIK cell culture bottles together, mixed culture.
6, amplification cultivation
(1) Day8: microscopically observation cell state, cell mass quantity increase, and cell quantity increases, and culture medium color becomes It when being orange, is blown and beaten in Biohazard Safety Equipment with pipette and mixes cell, cell culture bags are connected by syringe sleeve, it will be thin Cell suspension in born of the same parents' culture bottle is transferred in cell culture bags by syringe sleeve.With the fresh IL-2's containing 1000IU/ml Serum free medium washs 2 culture bottles, and after washing culture medium is incorporated into cell culture bags, remaining addition residual body is added Long-pending heat inactivate and be centrifuged after autologous plasma, and add the serum free medium of the fresh IL-2 containing 1000IU/ml of 800ml；
(2) Day11: after three days, culture medium color obviously turns yellow, and cell quantity increased significantly, and it is new to add 1000ml at interval The fresh serum free medium that 1000IU/ml IL-2 has been added, total volume is 2L in culture bag, operation ibid step Day8, and right Cultivating system does microorganism detection, and testing result shows that cell is pollution-free；
(3) it is added in cell culture bags with the PD-1 factor that 1ml syringe draws 5mg~10mg.
7, cell is collected
Day15:
(1) cell culture bags are taken out from incubator, are placed in Biohazard Safety Equipment, by cell suspension from cell culture bags It is transferred in several centrifuge tubes, tightens lid, trim is placed on centrifugation in centrifuge and abandons supernatant；
(2) it washs for the first time: physiological saline is added, cell in centrifuge tube is resuspended, cell suspension in centrifuge tube is merged into three Pipe, sample detection endotoxin are centrifugation abandoning supernatant after feminine gender；
(3) it washs for second: physiological saline is added, cell in centrifuge tube is resuspended, cell suspension in centrifuge tube is merged into one Pipe is tightened after lid shakes up, assigns to two centrifuge tubes.Respectively plus physiological saline is to 50ml, shakes up wherein a pipe tightens lid, It draws cell sample and carries out cell count, survival rate test and flow cytometer detection, average cell number 1.12*10 in centrifuge tube10, put down Equal motility rate is 93%, is all larger than professional standard.Supernatant is abandoned in centrifugation；
(4) third time is washed: physiological saline is added, cell in centrifuge tube is resuspended, cell suspension is incorporated to a centrifuge tube In, supernatant is abandoned in centrifugation；
(5) cell packs: taking new physiological saline 10ml that cell in centrifuge tube is resuspended, and the 1ml that keeps sample is stored in -20 DEG C of ice Cell suspension is crossed cell screen clothes by case, and with sieve is crossed after other 10ml salt water washing centrifuge tube again, carefully by gained after filtering Dysuria with lower abdominal colic moves to cell and feeds back in bag, in addition adds human serum albumin 5ml, and the physiological saline fed back in bag is supplemented to particular volume Product, sealing, as cell final formulation.
The detection of two cell phenotype of embodiment
DC-CIK cell that the first cell factor to the 5th cell factor that the embodiment of the present invention utilizes is prepared with The existing DC-CIK cell obtained using A jacket cell factor culture carries out cell phenotype detection.
The subgroup that CIK cell mainly has killing activity by two kinds forms, NKT cell (CD3+CD56+) and CTL cell (CD3 + CD8+), wherein NKT cell is the main effects cell of CIK cell.
Professional standard are as follows: CD3+CD56+ cell proportion is not less than 70% not less than 10%, CD3+.
As a result as shown in Figure 1, it is 19.3%, CD3+ thin that the cell phenotype of A nested factor culture, which is CD3+CD56+ cell proportion, Born of the same parents' ratio is that 83.6%, CD3+CD8+ cell proportion is 72.3%.Preparation method provided by the invention, after cell culture 14 days It is 81.8%, CD3+ cell proportion is 89.5%, CD8+ that CD3+CD56+ cell proportion, which is 29.8%, CD3+CD8+ cell proportion, Not less than 40%.The result shows that DC-CIK cell effective cell content made from preparation method provided by the invention is more, and kill Active high, cell phenotype is higher than Industry code requirements.
Three mtt assay of embodiment detection DC-CIK cell kills ratio of outflow
It is effector cell, human lung carcinoma cell line (A549 cell) and human hepatoma cell strain (Huh7 cell) with DC-CIK cell For target cell, ratio is inoculated in 96 orifice plates by 5:1 respectively, and experiment component adds PD-1 group and PD-1 group is not added, and every group 3 multiple Hole.37 DEG C, cultivate 24 hours in 5% carbon dioxide cell incubator.20 μ l of MTT, 37 DEG C, the training of 5% carbon dioxide is added in every hole After supporting case standing 4h, culture medium is sucked out, 150ul DMSO is added in every hole, shakes 10min, dissolution to be precipitated.Then in microplate reader OD value is surveyed at upper 490nm, calculates killing rate.Take the average value of 4 test results.
The calculation formula of killing rate are as follows: killing rate (%)=[1- (effect target cell group OD value-effector cell organizes OD value)/(target Groups of cells OD value-blank control group OD value)] × 100%.
By Fig. 2 and Fig. 3 it is found that the killing activity for not adding the PD-1 factor that joined the DC-CIK cell of PD-1 is high.
The above, the above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although referring to before Stating embodiment, invention is explained in detail, those skilled in the art should understand that: it still can be to preceding Technical solution documented by each embodiment is stated to modify or equivalent replacement of some of the technical features；And these It modifies or replaces, the spirit and scope for technical solution of various embodiments of the present invention that it does not separate the essence of the corresponding technical solution.

Claims (10)

1. a kind of DC-CIK cell culture kit characterized by comprising DC cell non-serum culture medium and CIK cell without Blood serum medium；
DC cell non-serum culture medium includes the first cell culture factor and the second cell culture factor, the training of CIK cell serum-free Feeding base includes: the third cell culture factor, the 4th cell culture factor and nutrients；
First cell factor includes: interleukin-4 (IL-4) and the cell colony stimulating organism factor (GM-CSF)；
Second cell factor includes: IL-4, GM-CSF and tumor necrosis factor α (TNF-α)；
The third cell factor includes: interferon gamma (IFN-γ)；
4th cell factor includes: CD3pure, interleukin 1 α (IL-1 α) and interleukin 2 (IL-2).
2. DC-CIK cell culture kit according to claim 1, which is characterized in that in first cell factor The concentration of IL-4 is 16.7ng/ml~33.4ng/ml, and the concentration of GM-CSF is 60ng/ml~120ng/ml.
3. DC-CIK cell culture kit according to claim 1, which is characterized in that in second cell factor The concentration of IL-4 is 83.5ng/ml~167ng/ml, the concentration of GM-CSF is 300ng/ml~600ng/ml, the concentration of TNF-α For 25ng/ml~50ng/ml.
4. DC-CIK cell culture kit according to claim 1, which is characterized in that in the third cell factor The concentration of IFN-γ is 2500IU/ml~5000IU/ml.
5. DC-CIK cell culture kit according to claim 1, which is characterized in that in the 4th cell factor The concentration of CD3pure is 250ng/ml~500ng/ml, the concentration of IL-1 α is 2.5ng/ml~5ng/ml, the concentration of IL-2 is 500IU/ml~1000IU/ml.
6. DC-CIK cell culture kit according to claim 1, which is characterized in that the nutrients is self blood Slurry, calf serum or autoserum.
7. a kind of using DC-CIK cell culture kit culture DC-CIK cell described in claim 1 to 6 any one Method, which comprises the following steps:
Step 1: after being cultivated to serum free medium of the peripheral blood mononuclear cells addition containing the first cell factor, being added Serum free medium containing the second cell factor is cultivated, and DC cell is obtained；
Step 2: after the serum free medium that the cell factor containing third is added in Xiang Suoshu peripheral blood mononuclear cells is cultivated, adding Enter nutrients and the serum free medium containing the 4th cell factor is cultivated, obtains CIK cell；
Step 3: the DC cell being co-cultured with the CIK cell, obtains DC-CIK cell.
8. the method according to the description of claim 7 is characterized in that nutrients is added in step 2 and containing the 4th cell factor After serum free medium is cultivated, before obtaining the CIK cell, further includes: the serum-free containing the 5th cell factor is added and trains Support base and the nutrients；
5th cell factor includes the IL-2 that concentration is 1000IU/ml~2000IU/ml.
9. also being needed the method according to the description of claim 7 is characterized in that step 3 obtained the DC-CIK cell before 1~2 day It is added programmed death receptor 1 (PD-1).
10. according to the method described in claim 9, it is characterized in that, the dosage of the PD-1 is 5mg~10mg.
CN201811340439.3A 2018-11-12 2018-11-12 A kind of DC-CIK cell culture kit and its cultural method Pending CN109402055A (en)

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Application publication date: 20190301