CN105087381A - 3D immune cell culture scaffold and preparation method thereof - Google Patents
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/14—Scaffolds; Matrices
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
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- A61L31/044—Collagen
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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Abstract
The invention disclose a 3D immune cell culture scaffold and a preparation method thereof, wherein the preparation method comprises the steps of acquiring collagen; dissolving the collagen by using 0.03 to 0.06 mol/L of acetic acid to prepare a collagen solution of which the mass fraction is 1 to 2 percent; freezing and forming the collagen solution to obtain a collagen block; vacuum drying the collagen block to obtain a collagen sponge; performing vacuum thermal crosslinking on the collagen sponge at the temperature of 100 to 110 DEG C. Compared with other common culture carriers, the preparation method of a collagen scaffold aims at the interstitial environment of DC cells in vivo, and adopts the collagen to prepare a flexible porous scaffold, the fiber viscidity of the collagen can improve the adsorption effect of a DC, so that the semi-adherent type DC has the ability of full adherence; the 3D immune cell culture scaffold prepared by utilizing the rehydration and the affinity of the collagen sponge has porous microporous environment, which can be used for fixing the cells, so that the contact of DC and CIK is improved.
Description
Technical field
The invention belongs to immunocyte culture technique field, be specifically related to a kind of 3D immunocyte and cultivate support and preparation method thereof.
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, provides a kind of 3D immunocyte to cultivate support and preparation method thereof.
In order to realize foregoing invention object, the technical scheme of the embodiment of the present invention is as follows:
3D immunocyte cultivates a preparation method for support, comprises the steps:
Obtain collagen protein;
By the acetate dissolution of described collagen protein with 0.03 ~ 0.06mol/L, make 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.
In the preparation process of above-mentioned collagen scaffold of the present invention, compare other common culture carrier, it is directed to DC cell interstitial environment in vivo, collagen protein is adopted to make mushy flexible support, the fiber viscosity of collagen protein can promote the adsorption effect of DC, thus makes the DC of half adherent type can have entirely adherent ability; And mushy micropore environment of having of support of preparation, can be used for fixing the effect of cell, cause cannot stablizing with CIK the deficiency contacted with flowing to avoid suspending in liquid culture in a large amount of CIK Dual culture; Thus promote contacting of DC with CIK.
The present invention also proposes a kind of 3D immunocyte directly prepared according to aforesaid method further and cultivates support.The support that this collagen fabric is integrally connected is overall, mushy micropore environment that support self has, 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; Carry out 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 the preparation method that a kind of 3D immunocyte cultivates support, comprises the steps:
S01, is dissolved in collagen protein in the acetum of 0.03 ~ 0.06mol/L, makes the collagen solution of massfraction 1 ~ 2%;
S02, by collagen solution freeze forming in mould, obtains collagen block;
S03, carries out vacuum-drying by collagen block, obtains collagen sponge body;
S04, carries out Vacuum Heat and is cross-linked, namely obtain collagen scaffold at 100 ~ 110 DEG C by collagen sponge body.
In the preparation process of above-mentioned collagen scaffold of the present invention, compare other common culture carrier, it is directed to DC cell interstitial environment in vivo, collagen protein is adopted to make mushy flexible support, the fiber viscosity of collagen protein can promote the adsorption effect of DC, thus makes the DC of half adherent type can have entirely adherent ability; And mushy micropore environment of having of support of preparation, can be used for fixing the effect of cell, cause cannot stablizing with CIK the deficiency contacted with flowing to avoid suspending in liquid culture in a large amount of CIK Dual culture; Thus promote contacting of DC with CIK.
Simultaneously, the material of support of the present invention is collagen protein, and collagen scaffold can provide similar even identical Growth of Cells microenvironment tissue-derived with it and cell communication, closer to tumor growth pattern for cultured cell in vitro, form the structure of analog inner tissue, play 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.
Wherein in above-mentioned steps S01, first raw material collagen protein dissolution being made solution, 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.
Further in step S02 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 S03, 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 S04, and affect this effect in thermal crosslinking treatment, also in steps in S03 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 S03 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 S04 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 S04 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 S04, 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 S04, 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.
Wherein, 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 S01 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.
Above-mentioned preparation method of the present invention, in vain for the feature of the immunocytes such as DC and CIK, adopts collagen protein to make solution, then freezing parallel vacuum lyophilization.Collagen protein after vacuum hydro-extraction is carried out heat cross-linking solidification by final step S04 again, forms the support entirety that collagen fabric is integrally connected.Mushy micropore environment that support self has, 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.Carry out 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.
The present invention cultivates on the basis of support preparation method at above-mentioned 3D immunocyte, and the 3D immunocyte also proposing further directly to be prepared by aforesaid method cultivates support.
Adopt the support that aforesaid method prepares, through performance test, flexility is relatively good, be highly susceptible to preserving and using, and the surface of culture vessel has and good is adhered fixed effect after wetting a little, substantially after fixing, can not Automatic-falling, this point than the surperficial affinity of existing polymer support and adhesivity good; And carry out its rehydration of rehydration experimental verification, it can adsorb the water yield of its weight 2 ~ 4 times, uses the microtexture of electron-microscope scanning support, and its void ratio is more homogeneous simultaneously.Fundamental property is seen, better than the support over-all properties of existing logical product.
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, highlight performance and the quality of cavernous body support activity inducement cultivation DC and CIK that the present invention utilizes this to prepare voluntarily simultaneously, be 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 is prepared:
S01, 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);
S02, 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;
S03, is placed in 40h freeze-drying at-20 DEG C, vacuum by the blocks of solid that step S02 obtains, obtains collagen sponge;
S04, by collagen sponge heat cross-linking 15h under 105 DEG C of vacuum, obtains the collagen scaffold needed for testing;
S05, after collagen scaffold water-wet, carefully adheres on the inner-wall surface of T75 culturing bottle and T175 culturing bottle and culture bag.
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:
S11, gets the blood after 100ml anti-freezing process, is joined by blood in 4 50ml sterile centrifugation tube respectively;
S12, 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;
S13, 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;
S14, 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;
S15, 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
S21, 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.
S22, 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;
At the same time when the CIK of T175 culturing bottle is cultured to 4d, in T175 culturing bottle, add amplification cultivation liquid B to 100ml;
S31, 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;
S32, 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;
S40, 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, scrapes the DC in T75 culturing bottle with cell scraper, joins during CIK cultivates 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. 3D immunocyte cultivates a preparation method for support, it is characterized in that, comprises the steps:
Obtain collagen protein;
By the acetate dissolution of described collagen protein with 0.03 ~ 0.06mol/L, make 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.
2. 3D immunocyte as claimed in claim 1 cultivates the preparation method of support, it is characterized in that, by described collagen solution freeze forming step, and freezing 1 ~ 2h at described freeze forming process is used in-20 ~ 30 DEG C.
3. 3D immunocyte as claimed in claim 1 or 2 cultivates the preparation method of support, and it is characterized in that, carried out in Vacuum Heat cross-linking step by described collagen sponge body at 100 ~ 110 DEG C, the time of described thermal crosslinking treatment is 15 ~ 18h.
4. 3D immunocyte as claimed in claim 1 or 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.
6. 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, temperature is-20 ~ 30 DEG C.
7. 3D immunocyte as claimed in claim 1 or 2 cultivates the preparation method of support, and it is characterized in that, obtain in described collagen protein step, described collagen protein is for becoming fibrous collagen.
8. 3D immunocyte as claimed in claim 7 cultivates the preparation method of support, it is characterized in that, described one-tenth fibrous collagen 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.
9. 3D immunocyte cultivates a support, it is characterized in that, the preparation method that described 3D immunocyte cultivates the 3D immunocyte cultivation support of support according to any one of claim 1 to 7 prepares.
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| CN116510065A (en) * | 2022-01-28 | 2023-08-01 | 江苏京森生物医药新材料科技有限公司 | Recombinant humanized III type collagen hemostatic sponge and application thereof |
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