CN105112372A - Immune cell culture method - Google Patents
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Abstract
The invention provides an immune cell culture method. The immune cell culture method includes: acquiring a DC (dendritic cell) and a CIK (cytokine-induced killer); producing a culture vessel with a 3D (three-dimensional) collagen cell culture scaffold; inoculating the DC into the culture vessel for culturing and subjecting the DC in culture to induced culture by induction activating agents; subjecting the CIK and the DC subjected to induced culture to co-culture in the culture vessel. The immune cell culture method has the advantages that according to the steps, a culture vessel is changed into the culture vessel with the 3D collagen cell culture scaffold, and the scaffold has a microporous environment intrinsically and has good reconstitution property and cell affinity intrinsically, thereby being capable of keeping or absorbing nutriments to form a cell culture nutriment bed, providing a better cell culture environment than other scaffolds and improving cell growth and cell proliferation capabilities; during co-culture of the DC and the CIK, contact probability of the DC with the CIK can be increased locally by retention capability of cells in pores, so that activity and quality of the cells cultured by the DC and the CIK are improved.
Description
Technical field
The invention belongs to 3D technical field of cell culture, be specifically related to a kind of immunocyte cultural method.
Background technology
DC-CIK is total in immunity, DC (dendriticcell, dendritic cell) mainly isolated from myeloid tissue, blood there is very strong capture antigen, the ability of antigen-presenting and secretion capacity, specificity antineoplastic immunity reaction strong lastingly can be induced; And CIK (cytokine induced kill cell) is a group foreign cell that in human peripheral, mononuclearcell obtains in vitro after cytokine profiles stimulates.It has, and multiplication capacity is strong, to kill tumor activity high and kill the feature that knurl spectrum is wide, clinical application untoward reaction is little, is more efficiently Tumor-cytotoxic efiect cell in adoptive immunotherapy.In DC-CIK combined immunization, DC cell and CIK cell are carried out Dual culture, make the maturation mutually both having promoted DC between the two, more can promote the propagation of ClK and strengthen the tumor-killing effect of CIK.
But in the Dual culture of above-mentioned DC and CIK, due to half attached cell that DC itself is separated in mononuclearcell (PBMC) vitro culture.When carrying out Dual culture at present, all that DC is directly attached on culture vessel, the growth atmosphere of culture vessel is not as original living environment, cause the growth of DC, maturation and the aspect such as proterties, immunocompetence expressed all can not reach the level of initial body environment, greatly have impact on the angtigen presentation effect that DC will possess.And due to the power of CIK function relevant with the specificity that the cell density of DC and its are offered, the competitive multiplication capacity of low, the contrary CIK of DC in-vitro multiplication ability is powerful, DC deficiency can be caused to cause with the touch opportunity of CIK low, thus the Antigen-presenting role of DC cannot be played, thus reduce the treatment effect of CIK.
Therefore, when above-mentioned culture environment can not meet DC cultivation in-vitro multiplication, usual technician uses in cultivation that the tubular fibre cultivating usual cell is cultivated, microcarrier is cultivated and micro-capsule culture method etc. promotes the environment cultivated, to make up the deficiency of simple Liquid culturing bottle; But, the totally different microenvironment in human body of container wall configuration that the monokaryon attached cell of this class of DC is adherent, in the process of cultivating, cell paste self is also easily by the damage of shearing force; More mainly, the direction of growth of cell is tending towards two dimension more, cause adopting above-mentioned training method DC cell and antigen contact chance still not enough, cell quality still cannot reach good expection.
Summary of the invention
Object of the invention process is to overcome the deficiency of cellar culture bottle culture environment in above-mentioned vitro culture, and the culture vessel providing a kind of 3D of employing collagen cell to cultivate support carries out the cultural method of DC and CIK immunocyte.
In order to realize foregoing invention object, the technical scheme of the embodiment of the present invention is as follows:
A kind of immunocyte cultural method, comprises the steps:
Obtain DC and CIK;
Preparation has the culture vessel that 3D collagen cell cultivates support;
DC is inoculated in described culture vessel and cultivates, and carry out inducing culture with induced activation agent in the process of DC cultivation;
In described culture vessel, Dual culture is carried out with CIK with the DC after inducing culture.
Aforesaid method step of the present invention, culture vessel is wherein changed into there is the culture vessel that 3D collagen cell cultivates support, by mushy micropore environment that this support has self, and self there is good rehydration and cellular affinity energy, can keep or absorb the material bed that nutriment forms cell cultures, there is provided and compare the better cell culture environment of other supports, improve the ability of Growth of Cells and propagation; Simultaneously in the process of DC and CIK Dual culture, the hold facility of hole inner cell can promote the contact probability of DC and CIK partially, promotes DC and CIK cultured cells activity and quality.
Embodiment
In order to make object 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 only in order to explain the present invention, be not intended to limit the present invention.
The embodiment of the present invention proposes a kind of immunocyte cultural method, comprises the steps:
S10, obtains DC;
S20, preparation has the culture vessel that 3D collagen cell cultivates support;
S30, is seeded to DC in the culture vessel of step S20 and cultivates;
S40, carries out in step S30, in the process of amplification cultivation, inducing DC with induced activation agent;
S50, adds CIK, carries out DC-CIK Dual culture in the culture vessel after step S40 induction.
Aforesaid method step of the present invention, the method of cultivating for existing usual DC and CIK, culture vessel is wherein changed into there is the culture vessel that 3D collagen cell cultivates support, by mushy micropore environment that this support has self, and self there is good rehydration and cellular affinity energy, can keep or absorb the material bed that nutriment forms cell cultures, provide and compare the better cell culture environment of other supports, improve the ability of Growth of Cells and propagation; Simultaneously in the process of DC and CIK Dual culture, the hold facility of hole inner cell can promote the contact probability of DC and CIK partially, promotes DC and CIK cultured cells activity and quality.
During concrete above-mentioned steps of the present invention is implemented, DC and CIK being used for carrying out immunity cultivation of step S10 acquisition can be prepared voluntarily with fresh Cord blood, or directly purchase can.Then carry out immunity to cultivate, the standoff container of tool that emphasis is prepared in step S20, wherein 3D collagen cell cultivates support is not commercially available product, needs to prepare voluntarily, and concrete step can with reference to carrying out as follows:
S21, is dissolved in collagen protein in the acetum of 0.03 ~ 0.06mol/L, makes the collagen solution of massfraction 1 ~ 2%;
S22, by collagen solution freeze forming in mould, obtains collagen block;
S23, carries out vacuum-drying by collagen block, obtains collagen sponge body;
S24, carries out Vacuum Heat and is cross-linked, namely obtain collagen scaffold at 100 ~ 110 DEG C by collagen sponge body;
S25, the collagen scaffold prepared by step S24 is fixed on the inner-wall surface of cell culture container.
First, first raw material collagen protein dissolution being made solution in above-mentioned steps S21, is in order to the thiozell of collagen protein can be made in the process preparing solution to redistribute, ensure again shaping after the quality of solid shape; Although the present invention adopts directly to be adopted by collagen protein in the early stage preparation of 3D support freezingly drain direct curing molding, also engineering carrier can be obtained; But carry out trickle electron-microscope scanning after the carrier that such mode obtains to observe, its microtexture is disorderly and overall and unstable, inner eggs white fiber connects and unstable, is not often also cultured at DC and ripely just decomposes fragmentation.And if the hole of support is excessive in cell cultures, so cell cannot docile well, if hole is little, and so can be dead because of the deficiency of space, nutriment after the short division several times of cell; So make the homogeneous formation solution of its fiber, the inhomogenous defect of hole after avoiding directly draining with after dissolving in the present invention.And, the raw material collagen protein of above-mentioned collagen scaffold itself can directly go to buy or prepare voluntarily, the collagen protein adopted in step S21 of the present invention preferably adopts into fibrous collagen, main one side is that source is wide, comprise type i collagen, II Collagen Type VI, type III collagen, XI Collagen Type VI, XXIV Collagen Type VI and XXVII Collagen Type VI, account for 90% of collagen sum.Compare into fiber collagen, the fibrocollagenous α-chain of non-one-tenth, both containing triple helical territory (collagen domain, COL), also containing non-triple helical territory (noncollagen domain, NC), structure is unfavorable for be cross-linked to form the homogeneous and stable support of hole.
Further in step S22 by collagen solution freeze forming in mould, obtain collagen block; And in this step, based on collagen concentration in the present invention and freezing requirement, adopt refrigerating process to control about-20 ~ 30 DEG C 1 ~ 2h.Adopt the mode of freeze-drying to obtain the collagen protein solidified, can begin to take shape the blank of support on the one hand, another object is to maintain protein molecular in follow-up crosslinked process and is unlikely to sedimentation, assembles etc.Wherein, it is to be noted that the employing of this mould is the employing carried out based on the shape of rack forming, according to the size of the culture vessel of required filling or the shape of different culture vessel, mould also can change accordingly, or can designed, designed.In the process of freeze forming, general control time 1 ~ 2h, avoids temperature excessively lowly to carry out freeze-drying further and generates that the shearing force of ice crystal is excessive causes the sex change injury more verified to thiozell.
After being solidified into bulk, in step S23, the collagen block formed after solidification being carried out vacuum-drying, obtaining collagen sponge body.By vacuum, the ice crystal in the collagen block freezed directly is carried out distilling thus removing in this step, carry out dry process under vacuum, can biological activity be kept, especially avoid the situation such as temperature-sensitive and oxidation to make it be destroyed.And carry out sublimation drying in vacuum freezing after, can be formed " skeleton ", can hold its shape after drying, volume is almost constant.The cavernous body formed after freeze-drying, rehydration is good, can be reduced into the state before freeze-drying by stream rapidly.When the Sponge characteristics of this rehydration is used to the process of liquid nutrient medium after making support, self can absorb the feed compositions in a lot of liquid nutrient medium, after poppet self just can as the material bed of cell, growth for cell provides comparatively comprehensively three-dimensional environment, and not only just simple adhesion.
And further for the effect of quality, owing to needing to control the degree of crosslinking of thiozell and the degree of mummification in above-mentioned steps S24, and affect this effect in thermal crosslinking treatment, also in steps in S23 just collagen block carry out vacuum-drying and obtain in collagen sponge body step, vacuum drying degree.Based on this purpose, in the present invention, the time of drying of step S23 controls 36 ~ 48h, makes dry process lyophilize degree, and does not proceed to the step of parsing-desiccation.Lyophilize 36 ~ 48h is adopted to be sublimation drying (non-parsing-desiccation) mode of water vapour by ice distillation wherein, a large amount of free water molecule that what this process can remove is in collagen block originally in solution, and the Bound moisture adsorbed with protein molecular can not be removed; Retain these Bound moisture to be conducive to preventing excessive mummification in subsequent thermal cross-linking process, thus ensure the quality of support.And if add long-time further and adjust temperature, carry out parsing-desiccation, what planar water can be a large amount of is parsed from adsorbed state, like this and be unfavorable for follow-up support quality.So in the present invention the period be 36 ~ 48h, and in vacuum drying process, keep the temperature of temperature and freeze forming substantially quite.
The cavernous body formed after vacuum-drying is carried out vacuum and is cross-linked by step S24 afterwards, by heat 100 ~ 110 DEG C high under vacuum, protein molecule is cross-linked by the dehydration contracting platform between the hydroxyl on amino-acid residue, amino, carboxyl.Vacuum is crosslinked is also oxidation cross-linked in order to avoid occurring, because commonly descend protein oxidation also can be cross-linked, be specially under oxygen (generally having air) condition, the oxidized form that sulfhedryl and sulfhedryl can occur protein molecule is cross-linked thus generates disulfide linkage.Although this oxidation cross-linked hardness that can increase the rear crosslinked albumen of solidification in a way, but greatly reduces and collapses performance, be unfavorable for follow-up rehydration, snappiness also can be short of to some extent.Be cross-linked so above-mentioned cavernous body is carried out Vacuum Heat in step S24 of the present invention, avoid oxidation cross-linked situation, thus ensure that the process of heat cross-linking is unlikely to the rehydration performance greatly reducing support.
Meanwhile, the enforcement repeatedly in the present invention and investigation, in the vacuum crosslinking Treatment of step S24, the degree of thermal crosslinking treatment can have influence on the direct performance of scleroproein.Heat cross-linking overlong time, thiozell mummification is too serious, and when being used further to tissue culture so, the irreversible degree of support self dehydration or sclerosis is too high, rehydration afterwards, surperficial affinity all can reduce in a large number, thus reduce the effect of support for cell cultures; So through checking repeatedly, the present invention, by process control 15 ~ 18h crosslinked for vacuum in step S24, obtains the collagen scaffold needed for testing, avoids the easy premature breakdown of the inadequate support of degree of crosslinking on the one hand, also can avoid the defect that mummification is serious.
After step S24 completes, be adhered fixed after support is soaked on the inner-wall surface of cell culture container, namely obtain the above-mentioned culture vessel with 3D collagen cell cultivation support of the present invention, then carry out immunocyte cultivation with this culture vessel.
Further, the culture vessel of what DC step S20 was prepared by step S30 have 3D collagen cell cultivates support carries out a large amount of vitro culture, and the substratum in the process of cultivating can adopt the immunocyte nutrient solution being suitable for DC growth to carry out; The new substratum changing heterogeneity can be avoided like this to cause the cataclysm of DC cell paste growing environment and cause the change of cell quality level, be more beneficial to and maintain active and HLA-II antigen.In culturing step of the present invention, be suitable for the nutrient solution of DC growth, through overtesting and contrast, the GM-CSF that the nutrient solution of the preferred employing of the present invention contains 60 ~ 100IU/ml gentamicin sulphate, 0.5 ~ 1.5%PPP, final concentration are 40 ~ 60ng/ml, final concentration are that the DMEM nutrient solution of the IL-4 of 450 ~ 550IU/ml carries out.
In the process that step S40 cultivates, induced activation agent is added in nutrient solution in good time and immune induction is carried out to DC, and will note in implementing the opportunity that the agent of DC induced activation is added, because according to the process of growth of cell cultures, the cell paste itself being in original cuiture can keep the essential property of original cell preferably, if normal cell then can still retain diploid number; And transit to the cell of Secondary Culture phase, its differentiation degree or change in shape can be deepened further, thus avoid the opportunity of induction delaying as far as possible to cell quality be passaged to change greatly after carry out, preferably in advance.Simultaneously, it is to be noted, owing to just can there is the effect of carrying out slowly-releasing in nutrient solution after absorption between above-mentioned DC induced activation agent two compositions, so the agent of DC induced activation needed both EGFR and aluminium adjuvant first to carry out preadsorption before adding to nutrient solution, be added to again in nutrient solution after completing.And if employing is directly added in nutrient solution respectively, aluminium adjuvant may adsorb in a large number to other compositions in nutrient solution, and cause the absorption of EGFR not enough, affect effect of EGFR induction.
After above-mentioned steps S40 of the present invention induces, can also by the DC TNF-α (TumorNecrosisFactor after step S40 activation induction, tumour necrosis factor) stimulate, DC maturation and offering property of stable antigen can be promoted further; Embodiment can adopt the TNF-α of suitable proportion to be added directly in DC nutrient solution and continue to cultivate.
Finally carry out DC cell after step S50 step S40 induces and CIK to state on the invention in container prepared by step S20 and proceed Dual culture.
The present invention is based on conventional when adopting common Erlenmeyer flask, culturing bottle or Boiling tube to carry out, even if in the culturing process such as more advanced employing tubular fibre cultivation, microcarrier cultivation and micro-capsule culture method, the container wall configuration that the monokaryon attached cell of this class of DC is adherent or all totally different microenvironment in human body of microenvironment, in the process of cultivating, cell paste self is also easily by the cultivation defect of the damage etc. of shearing force; More mainly, the growth of cell is tending towards two dimensional surface more, in the process of cultivation DC cell and antigen contact chance low, angtigen presentation efficiency is low; So in the scenario above, the present invention, in the process of above-mentioned cultivation, the container inner wall for cultivating arranges one deck collagen scaffold.And have test to show, MSCs (mesenchymal stem cells MSCs) is seeded on three-dimensional PET and cultivates, when inoculum density is minimum, amplification times is the highest, and maintain many differentiation potentials of its undifferentiated state and cell, all related impacts of factor such as cell amplification and brace aperture rate, surface-area, water-absorbent, Implantation Rate.
Therefore, for the mononuclearcell needing in the present invention to carry out cultivating and DC cell, the collagen scaffold of the above-mentioned three-dimensional that the present invention adopts collagenic material to make is placed on the inwall of culture vessel.Collagen scaffold can provide similar even identical Growth of Cells microenvironment tissue-derived with it and cell communication for cultured cell in vitro, closer to tumor growth pattern, forms the structure of analog inner tissue, plays its function; Thus realize the differentiation directional induction being both beneficial to various types of cells, be beneficial to again maintenance and the propagation of cytodifferentiation phenotype.The cultivation carrying out DC in said vesse is induced to after quantity and shape reach requirement, adds CIK and carries out Dual culture; In the process of cultivating, support self has good rehydration function can store more nutriment, and the place that the hole in support can stably provide DC with CIK to contact, to avoid the situation that DC and CIK cell contact rate in moving phase are low.
The understanding of those skilled in the art can be easier to for the ins and outs and process approach that make above-mentioned enforcement of the present invention and implement reference, the cavernous body 3D cell culturing bracket activity inducement simultaneously highlighting the collagen protein that the present invention utilizes this to prepare voluntarily cultivates performance and the quality of DC and CIK, is illustrated below by way of specific embodiment.
Embodiment 1
DC and the CIK co-cultured cell body of DC-CIK combined immunization is finally obtained based on the present embodiment, therefore following simultaneously integration by these two portions of DC and CIK is carried out, and the basic culture solution adopted in process is once the DMEM containing 80IU/ml gentamicin sulphate, and concrete culturing process is as follows:
(1) the T75 culturing bottle of 3D collagen scaffold of the present invention and T175 culturing bottle and culture bag (capacity 1.5L) is prepared:
S11, according to collagen protein: the mass ratio of acetum is the ratio of 1:80, is dissolved in the acetum of 0.04mol/L makes collagen solution by I-type collagen (buying from Hua Yan bio tech ltd, Shanghai);
S12, collagen solution vacuum defoamation is placed on mould (each 10mm of length and width, high 2mm, this specification is adopted to test in the present invention, and the needs that technician can cultivate according to reality in a large amount of preparation and effect are from Row sum-equal matrix, this specification need not be defined in) in ,-20 DEG C of freezing 1.5h become blocks of solid;
S13, is placed in 40h freeze-drying at-20 DEG C, vacuum by the blocks of solid that step S22 obtains, obtains collagen sponge;
S14, by collagen sponge heat cross-linking 15h under 105 DEG C of vacuum, obtains the collagen scaffold needed for testing;
S15, after collagen scaffold water-wet, carefully adheres on the inner-wall surface of T75, T175 culturing bottle and culture bag, for subsequent use.
Certainly in order to avoid being subjected to the pollution of other miscellaneous bacterias, the operations of culturing bottle and support can aseptically be carried out.
(2) DC is prepared:
S21, gets the blood after 100ml anti-freezing process, is joined by blood in 4 50ml sterile centrifugation tube respectively;
S22, by centrifuge tube after the centrifugal 10min of 300g, the blood plasma on upper strata takes out separately for the preparation of low platelet blood plasma (the immunocyte nutrient solution for the preparation of follow-up);
Lower floor after centrifugal, containing the throw out of hemocyte, is suspended mixed solution with the resuspended extremely often pipe 30ml of injection physiological saline;
S23, then get 4 50ml centrifuge tubes containing 15ml lymphocyte separation medium, is suspended slowly by step S12 the upper strata that mixed solution pipettor transfers to lymphocyte separation medium; Middle tunica albuginea layer is drawn in new 50ml centrifuge tube after centrifugal 20 minutes by 400g, and the tunica albuginea layer of gained is mononuclearcell;
Meanwhile, repeat 3 ~ 4 times with after the fluid infusion to 45ml of injection physiological saline, centrifugal 5 minutes of 300g, cleans mononuclearcell, the cell paste of collected by centrifugation mononuclearcell after having cleaned;
S24, the mononuclearcell 20ml basic culture solution obtained by step S13 is resuspended, and suspension is added to cultivation 2 ~ 3h in the T75 culturing bottle containing 30 collagen scaffolds, to not pour in another one T175 culturing bottle by attached cell afterwards, and supply the nutrient solution of T175 culturing bottle to 50ml with basic culture solution, and add 0.5mlPPP and 50ulIFN-γ (final concentration is 1000IU/ml) cultivation; Here it should be noted that in T175 culturing bottle the CIK obtained after carrying out selectivity cultivation in never adherent mononuclearcell;
S25, after attached cell is not removed, adds 20ml immunocyte nutrient solution A and carries out selection cultivation, can obtain the DC in adherent mononuclearcell in T75 culturing bottle;
Wherein, immunocyte nutrient solution A: the basic culture solution being the IL-4 of 500IU/ml containing 1%PPP, the final concentration GM-CSF that is 50ng/ml, final concentration.
(3) DC and CIK is cultivated respectively
S31, cultivates 2d by the T175 culturing bottle cultivating CIK in step S14, adds 20mL immunocyte nutrient solution B in T175 culturing bottle; Wherein, immunocyte nutrient solution B: containing final concentration be the IL-1 α of 100IU/ml, the final concentration anti-CD49d McAb that is 50ng/ml, the final concentration basic culture solution that is the IL-2 of 300IU/ml.
S32, by cultivating the T75 culturing bottle incubation time of DC in step S15 to 4d, carrying out half amount with immunocyte nutrient solution A and changing liquid;
Simultaneously when the CIK of T175 culturing bottle is cultured to 4d, in T175 culturing bottle, add amplification cultivation liquid B to 100ml;
S40, induction DC:
S41, prepare induced activation agent: (final concentration is 0.125% by the Alum adjuvant of the 50ug/mLEGFR (LifeTechnologies company of the U.S.) of 1mL and 1mL, for mass volume ratio, BrenntagBiosector company of Denmark) mixing, carry out preadsorption in 4 DEG C;
S42, induction: when the DC of T75 culturing bottle is cultured to 6d, adds the DC induced activation agent of carrying out in advance adsorbing in T75 culturing bottle, induce;
Meanwhile, the CIK in T175 culturing bottle is proceeded in culture bag and cultivates, add amplification cultivation liquid to 300ml; The amplification cultivation liquid adopted in this step is be the basic culture solution of the IL-2 of 300IU/ml containing 1%PPP, final concentration;
S43, after step S32 about induces 1d, in T75 culturing bottle, adds 20ulTNF-α (tumour necrosis factor, final concentration is 20ng/ml), and the final DC of promotion is ripe and stablize.
(4) DC-CIK Dual culture:
S50, adds TNF-α after mono-day in step S40, collects the DC in T75 culturing bottle, joins in the culture bag of CIK cultivation and carries out Dual culture, and add amplification cultivation liquid to 600ml; Collecting cell after 2d.
(4) in order to verify the progressive of the inventive method, need to identify the efficiency of the cell that it obtains, whether Authentication-Type is accurately true, and whether efficiency has lifting really, and due to the beneficial effect of the cultural method of DC cell be that DC engulfs the ability of offering antigen and strengthens, this effect does not have direct Testing index, can only reflect this effect indirectly by the result of the multiplication capacity of CIK after Dual culture and phenotype analytical, concrete:
1, count CIK with trypan blue staining, calculate its proliferation times.
2, get the induction CIK cell of 14 days, after PBS washs 2 times, respectively get 1 × 10
6/ 100 μ l are placed in falcon pipe, and add 20 μ l mouse-anti people fluorescein-labeled CD3, CD16, CD56, CD3/CD56 and Isotype control IgG1/IgG1 respectively, 4 DEG C of lucifuges hatch 30min, machine analysis on flow cytometer after PBS washs 2 times.
Wherein, the detection of above-mentioned every repeats 3 contrasts, finally gets result mean value; The average result of CIK proliferation times is 4 ~ 5x10
8, compare the 2 ~ 3x10 of common CIK
7multiplication capacity have significant lifting, and Phenotypic examination finally determine type really for CIK.
3, after the CIK testing external performance that above-mentioned Dual culture obtains, final tumor-killing ability test is carried out further:
Using cell of the present invention as experimental group, to organize with target cell group and effector cell and jointly form three control groups, get 96 well culture plates, in every hole, experimental group adds effector cell (CIK) and each 100 μ L of target cell (K562 cell), effect target is than being 25:1, and effector cell is 1.25 × 10
5individual, target cell is 5000; Target cell group adds target cell and each 100 μ L of nutrient solution; Effector cell's group adds effector cell and each 100 μ L of nutrient solution, and each group arranges 3 parallel multiple holes.Put 37 DEG C of incubators and hatch 4h, add MTT20 μ L, hatch 4h again, supernatant is abandoned after centrifugal, every hole adds dimethyl sulfoxide (DMSO) (DMSO) 150 μ L, every hole absorbance A is detected in enzyme-linked immunosorbent assay instrument (wavelength is 570nm), with following formulae discovery kill rate=[1-(A experimental port-A effect hole)/A Target cell wells] × 100% after abundant dissolving;
Kill rate after experimental group of the present invention finally calculates is 78.6% (repeating the mean number of 3 times), compare after usual DC-CIK cell induction cultivates 3 weeks, improve more than 10% according to effect target than the kill rate 55 ~ 65% for detecting during 15:1 ~ 20:1, therefore the better cytoactive of DC and CIK and tumor-killing effect prepared by the present invention can be described.And mainly, DC cell count than usually cultivating obtain many, illustrate application of the present invention have support after culture vessel, DC growth more more, support has good lifting effect really in cell cultures.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (9)
1. an immunocyte cultural method, is characterized in that, comprises the steps:
Obtain DC and CIK;
Preparation has the culture vessel that 3D collagen cell cultivates support;
DC is inoculated in described culture vessel and cultivates, and carry out inducing culture with induced activation agent in the process of DC cultivation;
In described culture vessel, Dual culture is carried out with CIK with the DC after inducing culture.
2. immunocyte cultural method as claimed in claim 1, is characterized in that, has the step that 3D collagen cell cultivates the culture vessel of support, comprising described in preparation:
Obtain collagen protein;
Described collagen protein is dissolved in the acetum of 0.03 ~ 0.06mol/L, makes the collagen solution of massfraction 1 ~ 2%;
By described collagen solution freeze forming, obtain collagen block;
Described collagen block is carried out vacuum-drying, obtains collagen sponge body;
Described collagen sponge body is carried out Vacuum Heat at 100 ~ 110 DEG C be cross-linked, obtain collagen scaffold;
Described collagen scaffold is adhered fixed on the inner-wall surface of culture vessel.
3. as claimed in claim 2 by described collagen solution freeze forming step, freezing 1 ~ 2h at described collagen solution freeze forming process is used in-20 ~ 30 DEG C;
And/or carried out at 100 ~ 110 DEG C in Vacuum Heat cross-linking step by described collagen sponge body, the time of described thermal crosslinking treatment is 15 ~ 18h.
4. 3D immunocyte as claimed in claim 2 cultivates the preparation method of support, it is characterized in that, is undertaken in vacuum drying step by described collagen block, and described vacuum-drying is that vacuum-sublimation is dry.
5. 3D immunocyte as claimed in claim 4 cultivates the preparation method of support, and it is characterized in that, in described vacuum-sublimation drying process, time of drying is 36 ~ 48h;
And/or temperature is-20 ~ 30 DEG C in described vacuum-sublimation drying process;
And/or obtain in described collagen protein step, described collagen protein is one or more in type i collagen, II Collagen Type VI, type III collagen, XI Collagen Type VI, XXIV Collagen Type VI and XXVII Collagen Type VI.
6. the 3D immunocyte as described in any one of claim 1 to 5 cultivates the preparation method of support, it is characterized in that, is inoculated in described culture vessel by DC and cultivate, and carries out in inducing culture step with induced activation agent in the process of DC cultivation,
Described induced activation agent is the mixture that small cell lung cancer specificity EGFR and aluminium adjuvant adsorb mutually.
7. immunocyte cultural method as claimed in claim 6, it is characterized in that, the mass ratio of described induced activation agent small cell lung cancer specificity EGFR and aluminium adjuvant is 2:1 ~ 8:1;
And/or described aluminium adjuvant is at least one in aluminium hydroxide, aluminum hydroxide gel, aluminum phosphate, Tai-Ace S 150, tschermigite and potassium alum.
8. the 3D immunocyte as described in any one of claim 1 to 5 cultivates the preparation method of support, it is characterized in that, is inoculated in described culture vessel by DC and cultivate, and carry out inducing culture step with induced activation agent in the process that DC cultivates after, also comprises:
With TNF-α, stimulation process is carried out to the DC after inducing culture.
9. the 3D immunocyte as described in any one of claim 1 to 5 cultivates the preparation method of support, it is characterized in that, inoculated by DC in described culture vessel and carry out in culturing step, the nutrient solution of employing is the DMEM containing 60 ~ 100IU/ml gentamicin sulphate, 0.5 ~ 1.5%PPP, the GM-CSF of 40 ~ 60ng/mL, the IL-4 of 450 ~ 550IU/ml.
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