CN105695405A - CIK (cytokine-induced killer) cell amplification method - Google Patents
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Abstract
The invention relates to a CIK (cytokine-induced killer) cell amplification method which comprises the following steps: acquiring a single karyocyte, and carrying out induced culture on the single karyocyte by using a CIK cell inducer until CIK cells are collected; and putting the CIK cells in a serum-free culture medium, and carrying out in-vitro amplification culture, wherein the serum-free culture medium contains PPP, IL-2, IL-12 and IL-27. The method can enhance the amplification efficiency and maturation rate of the CIK cells, can enhance the CD3<+> and CD56<+> cell counts, and can enhance the killing activity.
Description
Technical field
The present invention relates to biological technical field, particularly to a kind of CIK cell amplification method。
Background technology
Cellular immunotherapy be a kind of emerging, there is the brand-new antitumour treatments of significant curative effect, compensate for the drawback of traditional operation, radiotherapy, chemotherapy, it is acknowledged as a kind for the treatment of means active, the most rising in 21st century combined therapy of tumour pattern, the currently the only treatment means being hopeful complete tumors destroyed cell in the Ye Shi world。Immunocyte refers to participation immunne response or the cell relevant to immunne response, including lymphocyte, dendritic cell, Monocytes/Macrophages, granulocyte, mastocyte etc.。
CIK (cytokine-inducedkiller, Chinese name: the killing cell of [autogenous cell immunotherapy] cytokine profiles induction) it is the mononuclearcell a group foreign cell group that cultivates acquisition under the effect of CD 3-resisting monoclonal antibody and cytokine profiles, wherein CD3+, CD56+Lymphocyte is main effects cell。CIK can direct killing tumor cell and virus infected cell；Inducing apoptosis of tumour cell；Suppress or killing tumor cell by discharging a large amount of inflammatory cytokine。CIK cell has that growth rate is fast, kill tumor activity height, to kill tumor spectrum wide, multidrug resistant tumor cell is sensitive equally, to features such as normal bone marrow hematogenesis precursor cytotoxicity are little, it is that the tumor cell killing activity having now been found that is strong, it is suitable for a kind of desirably effector lymphocyte of clinical practice, but this effector lymphocyte is extremely rare in normal peripheral blood, is only 1%～5%。As can be seen here, with regard to immune cell therapy, how to obtain necessary requirement sufficient amount of, that immunocompetence is strong effector lymphocyte is to ensure that therapeutic effect。
CIK cell can by external evoked and breed in a large number, conventional CIK preparation method is to add in culture fluid by the PERIPHERAL BLOOD MONONUCLEAR CELL separated, carry out stimulating induction, a number of CIK cell of final acquisition, but the final CD3 obtained by adding cell growth factor+And CD56+CIK cell quantity and killing activity are all not ideal enough。
Summary of the invention
The technical problem to be solved is, there is amplification efficiency difference for CIK cell amplification in prior art, the defects such as cell killing activity is low, it is provided that one can improve CIK cell amplification efficiency, especially improve CD3+And CD56+CIK cell quantity, improves the CIK cell amplification method of killing activity。
The technical solution adopted for the present invention to solve the technical problems is: provides a kind of CIK cell amplification method, comprises the following steps:
Obtain mononuclearcell, adopt CIK cell induction liquid that described mononuclearcell is carried out inducing culture to gathering in the crops CIK cell；
Described CIK cell is placed in serum-free medium and carries out amplification in vitro cultivation；
Wherein, containing PPP, IL-2, IL-12 and IL-27 in described serum-free medium。
In CIK cell amplification method provided by the invention, in described amplification in vitro incubation, within every 2～3 days, add containing PPP, IL-2, IL-12 and IL-27 serum-free medium。
In CIK cell amplification method provided by the invention, described serum-free medium is AIM-V culture medium。
In CIK cell amplification method provided by the invention, in described serum-free medium, the volume fraction of PPP is 3～5%, and the concentration of IL-2 is the concentration that concentration is 800～1200IU/ml, IL-27 of 800～1200IU/ml, IL-12 is 5～30ng/ml。
In CIK cell amplification method provided by the invention, in described serum-free medium, the concentration of IL-27 is 15～25ng/ml。
In CIK cell amplification method provided by the invention, in described serum-free medium, the volume fraction of PPP is 4%, and the concentration of IL-2 is the concentration that concentration is 1000IU/ml, IL-27 of 1000IU/ml, IL-12 is 18ng/ml。
In CIK cell amplification method provided by the invention, described CIK cell derivant includes IFN-γ, anti-CD49d McAb and IL-1 α。
In CIK cell amplification method provided by the invention, in described CIK cell derivant, the concentration of IFN-γ be 800～1200IU/ml, anti-CD49d McAb the concentration that concentration is 10-150ng/ml and IL-1 α be 10-50 μ g/ml。
Implement CIK amplification method provided by the invention, it is possible to reach following beneficial effect: amplification efficiency and the maturing rate of CIK cell can not only be improved, and CD3 can be improved+And CD56+CIK cell quantity, improves killing activity。
Detailed description of the invention
CIK cell amplification method provided by the invention, comprises the following steps:
S1, acquisition mononuclearcell；
S2, with CIK cell derivant, mononuclearcell is carried out inducing culture 18～36 hours, to gathering in the crops primary CIK cell；
Wherein, CIK cell derivant includes IFN-γ (Interferon-γ, IFN-γ), anti-CD49d McAb and IL-1 α (Interleukin, interleukin-11 α)；Preferably, in derivant A, the concentration of IFN-γ is 800～1200IU/ml, and the concentration of anti-CD49d McAb is the concentration of 10-150ng/ml, IL-1 α is 10-50 μ g/ml；
Primary CIK cell is carried out amplification in vitro and cultivates 7～21 days by S3, employing serum-free medium, wherein, containing PPP (PoorPlateletPlasma in serum-free medium, platelet-poor plasma), IL-2 (Interleukin, interleukin-22), IL-12 (Interleukin, interleukin 12) and IL-27 (Interleukin, interleukin-22 7)；Within every 2～3 days, add containing PPP, IL-2, IL-12 and IL-27 serum-free medium, to keep PPP, IL-2, IL-12 and IL-27 concentration constant；Preferably, serum-free medium is AIM-V culture medium, is purchased from Gibco company of the U.S.。
IL-27, it is possible to remarkably promote the propagation of CIK cell, and initial CD4 can be induced+T cell expresses IL-12 beta 2 receptor so that it is obtain the reactivity to IL-12, and the collaborative IL-12 inducer T lymphocyte of IL-27 produces IFN-γ。
The peptide molecule that IFN-γ is had several functions by family forms, and has suppression virus replication, suppresses the effect such as tumor growth and immunomodulating, plays an important role in Cellular Immunity and humoral immunization。IFN-γ can express high-affinity IL-2R thus activating T cell by induced monocyte release cells factor IL-12, promotes the propagation of CIK cell。
And IL-27 combines IL-2 and can be obviously promoted CIK cell maturation, shorten CIK cell cultivation cycle, improve CIK cell and kill ability, promote the expression of the horizontal high expressed of CIK cell IFN-γ and TNF α (TumorNecrosisFactor, tumor necrosis factor-alpha)。
To sum up, IL-27 combines IL-12 and IL-2 cultivation can reduce the toxic and side effects that heavy dose of cytokine is brought on the one hand, can obtain the CIK cell of sufficient amount and function, especially CD3 on the other hand+And CD56+CIK；Meanwhile, it is capable to reduce the using dosage of cytokine。
PPP can provide certain nutritional support for cell, and can improve amplification rate and the Maturity of CIK cell, improves the activity of CIK cell。
Preferably, in serum-free medium, the volume fraction of PPP is 3～5%, and the concentration of IL-2 is the concentration that concentration is 800～1200IU/ml, IL-27 of 800～1200IU/ml, IL-12 is 5～30ng/ml；It is further preferable that the concentration of IL-27 is 15～25ng/ml。
In order to make the purpose of the present invention, technical scheme and advantage clearly understand, below in conjunction with embodiment, the present invention is further elaborated。Should be appreciated that specific embodiment described herein is only in order to explain the present invention, is not intended to limit the present invention。
Embodiment 1
CIK cell amplification method provided by the invention, comprises the following steps:
S1a, from peripheral blood, isolate mononuclearcell；
S11a, aseptic condition gather healthy volunteer peripheral blood 20ml, anticoagulant heparin, in GMP Clean Operating Lab, 20 DEG C, centrifugal 1000g × 10min, draws upper plasma and is placed in another aseptic 50ml centrifuge tube, and blood plasma is centrifuged in rear 56 DEG C of water baths to place 30min standby, add D-PBS buffer and the hemocyte layer dilution proportion with 1:1, piping and druming mixing。
S12a, employing Ficoll density－gradient centrifuga－tion method separation human peripheral blood single nucleus cell；
The lymphocyte separation medium of 25ml is filled respectively with two 50ml centrifuge tubes, slowly along centrifuge tube tube wall, the blood after dilution is added to lymphocyte separation medium upper strata with Sterile pipette, the volume ratio forming obvious interface, dilute blood and lymphocyte separation medium is 1:1。
S13a, high speed low temperature centrifugal machine, 20 DEG C, centrifugal, 800g × 30min；
In S14a, centrifugal rear centrifuge tube, liquid is divided into four layers, is red blood cell layer, lymphocyte separation medium layer, tunica albuginea layer and plasma layer from down to up respectively。Namely PERIPHERAL BLOOD MONONUCLEAR CELL is positioned at tunica albuginea layer, is moved in another 50ml sterile centrifugation tube by this layer with Sterile pipette。
S15a, washing tunica albuginea confluent monolayer cells three times, fill it up with 50ml with D-PBS solution, fully mixing tunica albuginea confluent monolayer cells, 20 DEG C, centrifugal: 1000g × 10min, abandoning supernatant, it is thus achieved that PERIPHERAL BLOOD MONONUCLEAR CELL, repeated washing three times。
After S16a, centrifuge washing, sopping up supernatant, by GT-T551 culture medium by cell piping and druming mixing, a small amount of cell of sucking-off counts, and adjusting cell concentration is 1 × 106Individual/ml, plants in, in the coated 175cm culture bottle of CD3-Ab and FN, being added the appropriate autoserum separated, for CIK cell in vitro inducing culture。
S2a, PERIPHERAL BLOOD MONONUCLEAR CELL is carried out inducing culture to changing into CIK cell；
The mononuclearcell obtained in step S1a is resuspended in AIM-V culture medium, and adds CIK cell derivant, be placed in 37 DEG C, 5%CO2Incubator is cultivated 24 hours；Wherein in CIK cell derivant, the concentration of IFN-γ is 1000IU/ml, and the concentration of anti-CD49d McAb is the concentration of 80ng/ml, IL-1 α is 30 μ g/ml；
S3a, CIK cell is carried out amplification in vitro cultivation；
The AIM-V culture medium of the IL-27 of IL-12 and the 18ng/ml of IL-2,1000IU/ml of containing PPP, 1000IU/ml that volume fraction is 4% is added in CIK cell, within every 2～3 days, add containing PPP, IL-2, IL-12 and IL-27 fresh culture, cultivate 21 days。
Wherein, the acquisition process of PPP is: take the peripheral blood after 100ml anticoagulant processes, peripheral blood is added separately in the sterile centrifugation tube of 4 50ml, 300g is centrifuged 10min, being transferred to by all of upper plasma in 2 new sterile centrifugation tube, 56 DEG C of water-bath inactivation 30min, after the centrifugal 10min of 400g, collecting upper strata yellow liquid and PPP, 4 DEG C store for future use。
With method for resuscitation, there is significant beneficial effect for verifying that microencapsulation immunocyte provided by the invention is frozen further, following experiment is set and is verified。
CIK cell after detection group 1 amplification that the corresponding embodiment of the present invention 1 obtains respectively；
The CIK cell that matched group is obtained by existing CIK cell amplification method, particularly as follows: the PERIPHERAL BLOOD MONONUCLEAR CELL serum-free medium of collection is adjusted concentration is 1 × 106U/ml, the IFN-γ adding 1000U/ml on the 1st day；
Subsequently culture fluid is placed in 37 DEG C, CO2Concentration is 5%, humidity be 100% constant incubator in cultivate 24 hours；It is subsequently added IL-1 α, IL-2 and CD3 monoclonal antibody so that IL-1 α, IL-2 and CD3 monoclonal antibody final concentration respectively 300U/ml, 1000U/ml and 500U/ml in the medium；
Then continue at 37 DEG C, CO2Concentration is 5%, humidity be 100% constant incubator in cultivate, every 3 days add culture medium, make cell concentration be maintained at 1 × 106The concentration of U/ml, IL-1 α and CD3 monoclonal antibody is kept at 300U/ml, 1000U/ml and 500U/ml。
1, the mensuration of cell proliferation multiple
Counting after the CIK cell Trypan Blue of acquirement with hematimeter, by current total cellular score divided by the mononuclearcell number before cultivating, numerical value is the amplification times of cell again。With the method can the proliferative conditions of dynamic observation of cell, concrete condition is in Table 1。
Table 1
| Cultivated days | 0 | 7 | 14 | 21 |
| Detection group 1 | 1 | 24.38±4.33 | 108.36±5.46 | 175.10±7.42 |
| Matched group | 1 | 12.47±4.12 | 52.37±5.27 | 83.42±6.77 |
Testing result: result shows, along with the prolongation of incubation time, the increased times of two groups of cells is all improving, but the increased times that detection group is on each timing node is significantly higher than detection group, and the amplification times of two groups of cells all reached summit at 21 days。
2, two groups of iuntercellular phenotype analyticals compare
Taking two groups of CIK cell at the 0th, 7,14,21 days respectively, make cell suspension, adjusting cell concentration after washing is 1 × 105U/ml, add the monoclonal antibody of labelling, hatch in room temperature dark place 15 minutes, wash away Excess antibody, using flow cytomery, result is in Table 2.
Table 2
Testing result: along with the prolongation of incubation time, CD3+ cell and the ratio of CD3+CD56+ cell in two groups of cells are all increasing, and difference between the two is not statistically significant；CD3+CD4+ cell proportion in two groups of cells reduces as time went on, and from the 7th day, the difference between two groups was statistically significant；CD3+CD56+ cell proportion aspect in two groups of cells, detection group is above matched group and difference statistically significant (P < 0.01) at each timing node。This shows that both of which can be greatly improved the ratio of CD+ cell, but in the CD3+CD56+ cell proportion in CIK cell, detection group to be substantially better than matched group。
3, mtt assay tests two groups of cells in vitro killing activities
Taking the logarithm the K562 tumor cell line of trophophase, re-suspended cell concentration is 1 × 105Individual/ml, 5 × 104Individual/ml, 2.5 × 104Individual/ml, every hole 100 μ l is laid in 96 level land, hole plates, is placed in 37 DEG C, CO2Concentration is 5%, humidity be 100% constant incubator in cultivate 24 hours stand-by；
It is 1 × 10 by cultivating two groups of CIK cell of 14 days resuspended6Individual/ml, add in aforementioned 96 orifice plates, make effect target than for 10:1,20:1 and 40:1, each concentration respectively sets 4 multiple holes, and set two groups of CIK cell not reacting with tumor cell as effector lymphocyte's blank, after co-culturing 48 hours, every hole adds MTT solution (5mg/ml) 20 μ l, after continuing cultivation 4 hours, being centrifuged and abandon supernatant, every hole adds DMSO100 μ l, measures absorption values in 570nm place, calculate killing rate, killing rate computing formula: killing rate=[1-(test hole A value-effector lymphocyte's A value)/target cell control wells A value] × 100%
Table 3
Testing result: under different effect targets is compared to and uses, the killing rate of tumor cell line is above matched group by detection group, and the difference between different effect target ratios is statistically significant, imitates target than under condition in difference, the killing rate of experimental group is substantially better than matched group, and difference is statistically significant。
Above embodiments of the invention are described; but the invention is not limited in above-mentioned detailed description of the invention; above-mentioned detailed description of the invention is merely schematic; rather than it is restrictive; those of ordinary skill in the art is under the enlightenment of the present invention; without departing under present inventive concept and scope of the claimed protection situation, it may also be made that a lot of form, these belong within the protection of the present invention。
Claims (8)
1. a CIK cell amplification method, it is characterised in that comprise the following steps:
Obtain mononuclearcell, adopt CIK cell derivant that described mononuclearcell is carried out inducing culture to gathering in the crops CIK cell；
Described CIK cell is placed in serum-free medium and carries out amplification in vitro cultivation；
Wherein, containing PPP, IL-2, IL-12 and IL-27 in described serum-free medium。
2. CIK cell amplification method according to claim 1, it is characterised in that in described amplification in vitro incubation, within every 2～3 days, add containing PPP, IL-2, IL-12 and IL-27 serum-free medium。
3. CIK cell amplification method according to claim 2, it is characterised in that described serum-free medium is AIM-V culture medium。
4. CIK cell amplification method according to claim 3, it is characterised in that in described serum-free medium, the volume fraction of PPP is 3～5%, the concentration of IL-2 is the concentration that concentration is 800～1200IU/ml, IL-27 of 800～1200IU/ml, IL-12 is 5～30ng/ml。
5. CIK cell amplification method according to claim 4, it is characterised in that in described serum-free medium, the concentration of IL-27 is 15～25ng/ml。
6. CIK cell amplification method according to claim 4, it is characterised in that in described serum-free medium, the volume fraction of PPP is 4%, and the concentration of IL-2 is the concentration that concentration is 1000IU/ml, IL-27 of 1000IU/ml, IL-12 is 18ng/ml。
7. CIK cell amplification method according to claim 1, it is characterised in that described CIK cell derivant includes IFN-γ, anti-CD49d McAb and IL-1 α。
8. CIK cell amplification method according to claim 7, it is characterised in that described CIK cell derivant, the concentration of IFN-γ be 800～1200IU/ml, anti-CD49d McAb the concentration that concentration is 10-150ng/ml and IL-1 α be 10-50 μ g/ml。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112029723A (en) * | 2020-09-18 | 2020-12-04 | 郑州佐爵生物科技有限公司 | Method for culturing umbilical cord blood CIK cells in vitro |
| WO2024103868A1 (en) * | 2022-11-16 | 2024-05-23 | 南京鼓楼医院 | Staged culture method for in-vitro expansion of human cik cells and use thereof |
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