CN105441390A - In-vitro three-dimensional amplification culture method for NK cells - Google Patents
In-vitro three-dimensional amplification culture method for NK cells Download PDFInfo
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Abstract
The invention relates to an in-vitro three-dimensional amplification culture method for NK cells. The culture method comprises the following steps: obtaining NK cells; inoculating a three-dimensional culture bracket with the NK cells; and performing in-vitro three-dimensional amplification culture, wherein the three-dimensional culture bracket is a chitosan-sodium alginate bracket. Through the porous structure as well as the mechanical properties and biocompatibility of the chitosan-sodium alginate bracket, the growing space of the NK cells and the contact area with nutrient substances are enlarged, and the NK cells can be uniformly distributed in the chitosan-sodium alginate bracket, thus the cell activity of the NK cells can be improved, and the improvement of the amplification rate and killing activity of the NK cells is facilitated; and the defects such as low activity and low amplification rate of NK cells caused by culturing the NK cells with feeder cells in a two-dimensional environment in the prior art are solved.
Description
Technical field
The present invention relates to biotechnology and biomedicine field, particularly a kind of external three-dimensional amplification cultivation method of NK cell.
Background technology
Natural killer cell (Naturalkillercells, NK cell), as a large quasi-lymphocyte colony arranged side by side with T cell, B cell, is the important member of natural immune system.NK cell, except participating in innate immune response, also plays important regulating and controlling effect in the immunomodulatory of adaptive immunity.The target cell of NK cell mainly contains some tumour cell (comprising part clone), virus infected cell, some autologous tissue's cell (as hemocyte), parasite etc., therefore, NK cell is antitumor, the anti-infectious important immune factor of body, also participates in the IIth type allergy and graft-vs-host reaction.To sum up, the key of clinical frequent application is promoted when the in vitro amplification of NK cell and activation, and the amplification method of NK cell is separated NK precursor or CD56 and uses the steps such as feeder cell before being mainly included in two dimension cultivation at present, not only need feeder cell, operation more loaded down with trivial details, and the expanding effect of NK cell is not obvious, and the NK cell killing activity obtained is poor.
Summary of the invention
Technical problem to be solved by this invention is, there is the defects such as NK cell amplification rate low and killing activity difference for the prior art NK cell that increases in two-dimensional environment, a kind of external three-dimensional amplification cultivation method that can improve the NK cell of NK cell amplification rate and killing activity is provided.
The technical solution adopted for the present invention to solve the technical problems is: a kind of external three-dimensional amplification cultivation method providing NK cell, and described cultural method comprises the following steps:
Obtain NK cell;
Described NK cell is inoculated in dimensional culture support and carries out external three-dimensional amplification cultivation; Wherein, dimensional culture support is chitin-sodium alginate support.
In the external three-dimensional amplification cultivation method of NK cell provided by the invention, the preparation process of described chitin-sodium alginate support, comprises the following steps:
Getting sodium alginate is dissolved in distilled water, and chitosan is dissolved in acetum, heats swelling; Regulate pH to 3.0 ~ 5.0 of chitosan and sodium alginate soln respectively with Glacial acetic acid after, mix, in-5 DEG C after vacuum defoamation--freezing and freeze-drying at 198 DEG C.
In the external three-dimensional amplification cultivation method of NK cell provided by the invention, the preparation process of described chitin-sodium alginate support, after being also included in chitosan and sodium alginate soln mixing, adds the step of the divalent-metal ion salts solution except magnesium ion.
In the external three-dimensional amplification cultivation method of NK cell provided by the invention, obtain the process of NK cell, comprise the following steps:
Obtain CD34
+hemopoietic stem cell carries out stimulation in containing the stem cell media of hematopoietic stem cell growth factor and is divided into NK cell.
In the external three-dimensional amplification cultivation method of NK cell provided by the invention, described hematopoietic stem cell growth factor comprises interleukin-2, IL-12, interleukin-15 and IL-18.
In the external three-dimensional amplification cultivation method of NK cell provided by the invention, in described stem cell media, the concentration of interleukin-22 is the concentration that the concentration of 100-300U/ml, IL-12 is 0.5-4.5ng/ml, the concentration of interleukin-15 is 2-8ng/ml and IL-18 is 5-15ng/ml.
In the external three-dimensional amplification cultivation method of NK cell provided by the invention, described hematopoietic stem cell growth factor comprises interleukin-2, and is also added with CD 3-resisting monoclonal antibody in described hematopoietic stem cell growth factor.
In the external three-dimensional amplification cultivation method of NK cell provided by the invention, in described stem cell media, the concentration of interleukin-2 is 5-15ng/ml, the concentration of CD 3-resisting monoclonal antibody is 5-15ng/ml.
In the external three-dimensional amplification cultivation method of NK cell provided by the invention, the step of described NK cell being carried out to amplification in vitro cultivation is: ground to add in acetic acid by described chitin-sodium alginate and redissolve, and add the described stem cell media containing hematopoietic stem cell growth factor, inoculate described NK cell and carry out cultivation 20-50 days.
In the external three-dimensional amplification cultivation method of NK cell provided by the invention, described CD34
+the acquisition process of hemopoietic stem cell, comprises the following steps:
From hemocyte, isolate mononuclearcell, go out CD34 through magnetic bead sorting
+hemopoietic stem cell.
Implement NK cell expansion ex vivo cultural method provided by the invention, following beneficial effect can be reached: the present invention carries out amplification cultivation by adopting chitin-sodium alginate support to NK cell, utilize the multi-pore structure of chitin-sodium alginate support and mechanical property thereof and biocompatibility, expand the growing space of NK cell and the contact area with nutritive substance, also NK cell can be made can be evenly distributed in chitin-sodium alginate support, avoid cell aggregation agglomerating, promote the contact of NK cell and nutritive substance, thus the cytoactive of NK cell can be improved, be conducive to the amplification rate and the killing activity thereof that improve NK cell, thus solve in prior art the microenvironment of adopt feeder cell to cultivate in two-dimensional environment NK cytoactive that NK cell exists is poor, amplification rate is low etc. defects simulation NK Growth of Cells.
Embodiment
It is low to there is amplification rate in the mode of carrying out amplification cultivation for solving NK cell in prior art in two-dimensional environment, the defects such as NK cell killing activity difference, innovative point of the present invention is the three-dimensional amplification cultivation mode providing a kind of NK cell, by utilizing the multi-pore structure of chitin-sodium alginate support and mechanical property thereof and biocompatibility, expand the growing space of NK cell and the contact area with nutritive substance, also NK cell can be made can be evenly distributed in chitin-sodium alginate support, thus the cytoactive of NK cell can be improved, be conducive to the amplification rate and the killing activity thereof that improve NK cell, thus solve in prior art adopt feeder cell to cultivate in two-dimensional environment NK cytoactive that NK cell exists is poor, amplification rate is low etc. defect.
Particularly, the external three-dimensional amplification cultivation method of NK cell provided by the invention, comprises the following steps:
S1, acquisition NK cell;
S2, NK cell is inoculated in dimensional culture support and carries out external three-dimensional amplification cultivation; Wherein, dimensional culture support is chitin-sodium alginate support.
Further, in step sl, NK cell can come from peripheral blood, Cord blood, decidua tissue and/or lymphoglandula.NK cell surface expression CD56 antigen, does not express CD3 antigen, and the difference according to CD56 antigen is expressed, and NK cell can be divided into CD56
dimcD16
brightand CD56
brightcD16
dim/negtwo large subgroups; CD56
brightcD16
dim/negnK cell is then mainly present in lymphoglandula and decidua tissue, and with secrete cytokines if interferon-γ is for major function, killing ability is more weak; And CD56
dimcD16
brightnK cell is then mainly present in peripheral blood, has pore-forming protein in abundant born of the same parents, and killing ability is comparatively strong, therefore, in the present invention, preferentially adopts from peripheral blood, obtains NK cell.
NK cell is mainly by CD34
+hemopoietic stem cell differentiation and development under the stimulation of hematopoietic stem cell growth factor is ripe NK cell, and the etap of NK cell is: from CD34
+hemopoietic stem cell is first divided into common lymphoid sample precursor cells, common lymphoid sample precursor cells regeneration NK precursor cell, NK precursor cell is grown for immature NK cell, finally, immature NK cell obtains reactivity and Inhibitory receptor, and obtains the NK cell that functional development is maturation.
Further, obtain the process of NK cell, comprise the following steps: obtain CD34
+hemopoietic stem cell is cultivated in containing the stem cell media of hematopoietic stem cell growth factor.
Wherein, for by CD34
+hemopoietic stem cell differentiation and development becomes the hematopoietic stem cell growth factor of NK cell, comprises interleukin-2, IL-12, interleukin-15 and IL-18; And preferably, in stem cell media, the concentration of interleukin-2 is the concentration that the concentration of 100-300U/ml, IL-12 is 0.5-4.5ng/ml, the concentration of interleukin-15 is 2-8ng/ml and IL-18 is 5-15ng/ml.
Interleukin-2 can induce CD34
+hemopoietic stem cell changes into NK cell, and through interleukin-2 induction CD34
+hemopoietic stem cell obtains NK cell and has obvious cytotoxicity, enhances the killing activity of NK cell; Meanwhile, interleukin-2 can also promote the propagation of NK cell, maintains the growth of NK cell long-period; And the activity of NK cell can be strengthened at short notice; IL-2 can also promote NK emiocytosis IFN-γ, increases it and expresses IL-2R+ subunit etc.
IL-12 can produce the cytokines such as IFN_ γ by induced NK cell, is conducive to promoting the growing multiplication of NK cell and active raising.
Interleukin 18 can strengthen the lytic activity of NK cell, activates the activity of NK cell, strengthens the cytotoxicity of NK cell, can promote again the killing activity that NK cell that pore-forming protein mediates is traditional to target cell.
Interleukin-15 can not only promote the reproduction restraint of hemopoietic stem cell, and can promote the expression of this Activating receptor of NK cell NKG2D, strengthens NK cell killing related genes as the expression of pore-forming protein, thus strengthens the killing ability of NK cell.
Under the stimulation of these somatomedins, hemopoietic stem cell quantity increases, and hemopoietic stem cell also changes along with breaking up its phenotype, grow commitment, IL-2/IL-15R-β cytokine receptor on stem cell, namely raising appears in the expression of CD122, and stem cell is divided into NK precursor cell thereupon, NK precursor cell has directed ability of growing for ripe NK cell, but NK precursor cell surface does not express CD56 antigen; Simultaneously, NK precursor cell is because expressing CD122 molecule and the rise of IL-2/IL-15 receptor β, so strengthen very responsive to the stimulation of interleukin-15, interleukin-15 can promote the growth of NK cell, interleukin-15 mainly comes from bone marrow microenvironment, therefore, IL-15 is adopted can to simulate the microenvironment of NK Growth of Cells better; In addition, IL-15 can make NK cell precursors increase and be divided into the ripe NK cell of expressing various Inhibitory receptor and Activating receptor.
In another embodiment provided by the invention, hematopoietic stem cell growth factor comprises interleukin-2, is wherein added with CD 3-resisting monoclonal antibody; Preferably, in stem cell media, the concentration of interleukin-2 is 5-15ng/ml, the concentration of CD 3-resisting monoclonal antibody is 5-15ng/ml.
Wherein, CD 3-resisting monoclonal antibody can not only promote the growing multiplication of NK cell, and can strengthen the cytotoxicity of NK cell, promotes the killing activity of the NK cell of pore-forming protein mediation.
Further, acquisition CD34 is also comprised in step S1
+the process of hemopoietic stem cell, and CD34
+hemopoietic stem cell is then isolate mononuclearcell from peripheral blood hemocyte after, through magnetic bead sorting out.CD34 is gone out through magnetic bead sorting from mononuclearcell
+the detailed process of hemopoietic stem cell is:
Get mononuclearcell, re-suspended cell density is 2 × (10
7~ 10
8) individual/m1, the ratio adding 80 ~ 150 μ L antibody with every ml cells suspension adds CD34 mixtures of antibodies, incubated at room 15 ~ 30 minutes; Then add the ratio of 50 ~ 100 μ L magnetic beads in every ml cells suspension, add CD34
+magnetic bead fully mixes, and after incubated at room, washing, after mixing cell, operates A below performing: cell is placed in magnetic field after 5 ~ 10 minutes, remove magnetic field, washing, mixing cell, is placed in magnetic field 5 ~ 10 minutes again, remove magnetic field, washing, repeat this operation A more than at least 3 times, can CD34 be gathered in the crops
+hemopoietic stem cell.
In step S2, carrying out to NK cell the substratum that vitro culture adopts is stem cell media, can with cultivation CD34
+the stem cell media that hemopoietic stem cell adopts is consistent; Preferably, this stem cell media is GT-T551 substratum, purchased from precious biotechnology (Dalian) company.
Further, in step S2, the preparation process of chitin-sodium alginate support, comprises the following steps:
Getting sodium alginate is dissolved in distilled water, and chitosan is dissolved in acetum, heats swelling respectively; Regulate pH to 3.0 ~ 5.0 of chitosan and sodium alginate soln respectively with Glacial acetic acid after, mix, in-5 DEG C after vacuum defoamation--freezing and freeze-drying at 198 DEG C; Preferably, the mass ratio of sodium alginate and chitosan is 5:3 ~ 3:5.
Further, after chitosan solution and sodium alginate soln mixing, be also added with the divalent-metal ion salts solution except magnesium ion, preferably, divalent-metal ion salts solution is CaCl
2solution; Wherein, chitosan is the natural polysaccharide obtained by chitin deacetylase base, has good biocompatibility and biodegradability, but the physical strength of chitosan is poor; And sodium alginate is the natural polysaccharide extracted from brown alga, its biocompatibility is good, has good mechanical property and rack forming ability, better mechanical property; Therefore the chitin-sodium alginate support be compounded to form by sodium alginate and chitosan can form complementation in mechanical property and controlled degradation, not only increase the biocompatibility of support, and improve its mechanical property and porosity, can for a long time for NK cell provides three dimensional growth space, to simulate the microenvironment of NK cell growth in vivo, make NK cell fully can contact nutritive substance, finally reach the object of cytoactive, amplification rate and the killing activity improving NK cell.
Crosslinked and after losing rheological under the effect of the divalent-metal ion of sodium alginate beyond demagging, the flowing of water molecules receives suppression, and then it is high and have the gelinite of through hole to create water content, and the gelinite of sodium alginate is not thermal reversion, there is good stability and biocompatibility, therefore, after chitosan and sodium alginate Homogeneous phase mixing, add divalent-metal ion, chitosan can be surrounded wherein in the process that sodium alginate gel body is formed, make chitosan can be uniformly distributed in sodium alginate gel body, make the structure of dimensional culture support more firm, thus provide stable three dimensional growth space for NK cell, more be conducive to the tactophily of NK cell.
The detailed process of step S2 is:
Get the chitin-sodium alginate support after grinding, after redissolving in solubility promoter acetic acid, be inoculated in cell culture container, and add the stem cell media containing hematopoietic stem cell growth factor in cell culture container, the NK cell then obtained in inoculation step S1 is cultivated.
Further, the external three-dimensional amplification cultivation method of NK cell provided by the invention, the process steps more simplified is:
CD34 is isolated from peripheral blood
+hemopoietic stem cell;
Getting the chitin-sodium alginate support after grinding is dissolved in solubility promoter, be inoculated in cell culture container after fully dissolving, then add in the stem cell media containing hematopoietic stem cell growth factor in the cell culture container containing chitin-sodium alginate support; Wherein, hematopoietic stem cell growth factor comprises interleukin-2, IL-12, interleukin-15 and IL-18; Or hematopoietic stem cell growth factor is interleukin-2, wherein, is added with CD 3-resisting monoclonal antibody; Preferably, in stem cell media, the concentration of interleukin-2 is the concentration that the concentration of 100-300U/ml, IL-12 is 0.5-4.5ng/ml, the concentration of interleukin-15 is 2-8ng/ml and IL-18 is 5-15ng/ml; Or in stem cell media, the concentration of interleukin-2 is 5-15ng/ml, and the concentration of CD 3-resisting monoclonal antibody is 5-15ng/ml.
Again and inoculate CD34
+hemopoietic stem cell carries out cultivation 20-50 days, can gather in the crops the NK cell after amplification.
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.
Embodiment is:
Embodiment 1
The external three-dimensional amplification cultivation method of NK cell provided by the invention, comprises the following steps:
S1a, acquisition NK cell;
The separation of S11a, mononuclearcell;
Gather the blood sample of 100ml human peripheral, or the blood sample of 80mL umbilical cord blood, with the PBS solution dilution blood sample of 1-2 times of volume, after abundant mixing, obtain PBS blood sample mixed solution, then in each 50ml centrifuge tube, first add the Ficoll parting liquid of 15ml, the density of Ficoll parting liquid is 1.077g/mL; Slowly add the PBS blood sample mixed solution of 20-30ml again, room temperature is centrifugal, and whizzer lifting speed is all adjusted to 0, in 20 DEG C, 400g, and centrifugal 30min, middle layer is mononuclearcell; Carefully draw with pasteur pipet, use 1 × PBS to fill it up with centrifuge tube afterwards, abundant centrifuge washing mononuclearcell (400g, centrifugal l0min), re-suspended cell, for subsequent use.
S12a, CD34
+the sorting of hemopoietic stem cell;
By the mononuclearcell obtained in step S11a to be adjusted to 2 × 10 after Trypan Blue living cell counting
8the cell suspension of individual/m1, adds the ratio of 100 μ L antibody, adds CD34 mixtures of antibodies, blow and beat abundant mixing up and down with pipettor, incubated at room 15 minutes in every ml cells suspension; Then add the ratio of 50 μ L magnetic beads in every ml cells suspension, add CD34
+magnetic bead fully blows and beats mixing, cell, after 10 minutes, is proceeded to polystyrene tube (12 × 75rnm) by incubated at room, adds PBS washings (containing 2%FBS, 0.01%EDTA) to 2.5ml, 2 ~ 3 times are blown and beaten gently up and down in vitro, mixing cell with pipettor.Again test tube is inserted in magnetic pole, leave standstill five minutes; Then magnetic pole is picked up together with test tube, topple over magnetic pole to inversion state with continuous slow motion, pour out supernatant fraction.The cell of magnetic mark, due to the attraction of magnetic pole and magnetic field, is still stayed in vitro.Keep magnetic pole and test tube to be inverted 2 ~ 3 seconds, then make test tube mouth recovery position upwards.From magnetic pole, take out test tube, add 2.5ml washings, blow and beat cell suspension gently 2 ~ 3 times with pipettor, mixing cell, then by tube back magnetic pole, leave standstill five minutes.After repeated washing 4 times, that retain in test tube is CD34
+hemopoietic stem cell, for subsequent use.
S2a, by CD34
+hemopoietic stem cell is inoculated in dimensional culture support and cultivates;
The preparation of S21a, chitin-sodium alginate support
Getting 1.0g sodium alginate is dissolved in 100ml distilled water, getting 0.5g chitosan is dissolved in 1% acetum of 50ml, respectively by sodium alginate soln and chitosan solution after 50 DEG C of heating for dissolving swelling more than 8 hours, after sodium alginate and chitosan are fully dissolved, glacial acetic acid solution is added respectively in sodium alginate soln and chitosan solution, regulate pH value all to 3.0 of sodium alginate soln and chitosan solution, then, after being mixed with chitosan solution by sodium alginate soln, at the uniform velocity dripping massfraction is the CaCl of 3%
2solution, fully washs through deionized water, after vacuum defoamation, and at-20 DEG C freezing 1 hour, then freeze-drying 24 hours in freeze-dried machine.S22a, by CD34
+hemopoietic stem cell is inoculated in chitin-sodium alginate support and cultivates;
Get chitin-sodium alginate support that 0.5g grinds to be dissolved in 3% acetic acid of 10ml and 20ml distilled water and to redissolve, be then inoculated in 24 orifice plates containing GT-T551 substratum; Adjustment CD34
+the cell suspension density of hemopoietic stem cell is 1 × 10
9individual/ml, is inoculated in above-mentioned 24 orifice plates, does flow cytometer showed and detect the front CD34 of inoculation before inoculation
+hemopoietic stem cell content, and ensure CD34
+hemopoietic stem cell is more than 95%; Add interleukin-2, IL-12, interleukin-15 and IL-18 respectively again, and making in GT-T551 substratum, the concentration that the concentration of interleukin-2 is 200U/ml, the concentration of IL-12 is 2.5ng/ml and interleukin-15 is the concentration of 5ng/mL and IL-18 is 10ng/mL; In 37 DEG C, 5%CO
2incubator is cultivated, and first day half amount changes liquid once, within every two days afterwards, changes liquid once.
Embodiment 2
Be with the difference of embodiment 1, the concentration of the hematopoietic stem cell growth factor added in this embodiment in stem cell media is respectively: the concentration that the concentration of interleukin-2 is 100U/ml, the concentration of IL-12 is 0.5ng/ml and interleukin-15 is the concentration of 2ng/mL and IL-18 is 5ng/mL.
Embodiment 3
Be with the difference of embodiment 1, the concentration of the hematopoietic stem cell growth factor added in this embodiment in stem cell media is respectively: the concentration that the concentration of interleukin-2 is 300U/ml, the concentration of IL-12 is 4.5ng/ml and interleukin-15 is the concentration of 8ng/mL and IL-18 is 15ng/mL.
Embodiment 4
Be with the difference of embodiment 1, the hematopoietic stem cell growth factor added in this embodiment contains interleukin-2, is wherein added with CD 3-resisting monoclonal antibody; And in stem cell media: the concentration of interleukin-2 is the concentration of 5ng/ml and CD 3-resisting monoclonal antibody is 5ng/ml.
Embodiment 5
Be with the difference of embodiment 1, the hematopoietic stem cell growth factor added in this embodiment contains interleukin-2, is wherein added with CD 3-resisting monoclonal antibody; And in stem cell media: the concentration of interleukin-2 is the concentration of 15ng/ml and CD 3-resisting monoclonal antibody is 15ng/ml.
Embodiment 6
Be with the difference of embodiment 1, the hematopoietic stem cell growth factor added in this embodiment contains interleukin-2, is wherein added with CD 3-resisting monoclonal antibody; And in stem cell media: the concentration of interleukin-2 is the concentration of 10ng/ml and CD 3-resisting monoclonal antibody is 10ng/ml.
For verifying the unusual effect of the three-dimensional amplification cultivation method of NK cells in vitro provided by the invention further, carry out detection validation by following experiment.
Detected object:
Control group: be with the embodiment of the present invention 1 difference, the cell that this group adopts two-dimensional environment of the prior art to carry out the cultivation of NK cell amplification to obtain;
Test set 1-6: be respectively the cell that embodiment of the present invention 1-6 obtains.
1, the flow cytometry analysis of NK cell
Application BectonDickinson type flow cytometer counts the NK cell after amplification, and detected result is as following table 1.
Table 1
As can be seen from the above results, along with to CD34
+the increase of hemopoietic stem cell cultivated days, the cell quantity in test set 1-6 increases gradually, and all far above the cell quantity in control group; It can thus be appreciated that the NK cell quantity obtained by the external three-dimensional amplification cultivation method of NK cell provided by the invention is more, improves the amplification rate of NK cell.
2, the detection of NK cell killing activity
Collect the K562 cell (human erythorleukemia cell line K562 cell, buys) of logarithmic phase, be resuspended in 0.5ml containing in the RPMI1640 substratum of 10%FCS, by 100 μ Ci/L × 10
6cell adds 51Cr.In 37 ° DEG C, 5%CO
2hatch 2h in incubator, shake up once every 15min, wash 3 times after mark, adjustment cell concn to 1 × 10
5/ mL adds in round bottom 96 orifice plate, and 100 μ L/ holes, as the target cell in killing experiments.By 10:1,5:1 and 2.5:1 equivalence IE than the NK cell adding respective amount, at 37 DEG C, 5%CO
24h is hatched in incubator, 100 μ L supernatants are collected in every hole, survey gamma-rays cpm value, calculate kill rate as follows: kill rate (%)=(experimental group cpm value-Spontaneous release cpm value)/(maximum release cpm value-Spontaneous release cpm value) × l00%, detect data and see the following form 2:
Table 2
Detected result: from data in table 2, the cell killing rate of test set 1-6 is all greater than control group, illustrates thus, and the three-dimensional amplification cultivation method of NK cell provided by the invention improves the killing activity of NK cell.
In sum, in the external three-dimensional amplification cultivation method of NK cell provided by the invention, microenvironment in the body of chitin-sodium alginate simulation NK Growth of Cells is adopted to carry out amplification cultivation NK cell, growth and the amplification of NK cell can not only be promoted, improve cytoactive and the amplification rate of NK cell, but also the killing activity of NK cell can be improved, clinical application for NK cell has great importance, solve in prior art, complicated by the feeder cell operating process that amplification cultivation NK cell exists in two-dimensional environment, NK cell amplification rate is low, the problems such as killing activity difference.
More than be described in conjunction with embodiments of the invention; but the present invention is not limited to above-mentioned embodiment; above-mentioned embodiment is only schematic; instead of it is restrictive; those of ordinary skill in the art is under enlightenment of the present invention; do not departing under the ambit that present inventive concept and claim protect, also can make a lot of form, these all belong within protection of the present invention.
Claims (10)
1. an external three-dimensional amplification cultivation method for NK cell, is characterized in that: described cultural method comprises the following steps:
Obtain NK cell;
Described NK cell is inoculated in dimensional culture support and carries out external three-dimensional amplification cultivation; Wherein, dimensional culture support is chitin-sodium alginate support.
2. the external three-dimensional amplification cultivation method of NK cell according to claim 1, is characterized in that, the preparation process of described chitin-sodium alginate support, comprises the following steps:
Getting sodium alginate is dissolved in distilled water, and chitosan is dissolved in acetum, heats swelling; Regulate pH to 3.0 ~ 5.0 of chitosan and sodium alginate soln respectively with Glacial acetic acid after, mix, freezing and freeze-drying at-5 DEG C ~-198 DEG C after vacuum defoamation.
3. the external three-dimensional amplification cultivation method of NK cell according to claim 2, it is characterized in that, the preparation process of described chitin-sodium alginate support, after being also included in chitosan and sodium alginate soln mixing, adds the step of the divalent-metal ion salts solution except magnesium ion.
4. the external three-dimensional amplification cultivation method of NK cell according to claim 1, is characterized in that, obtains the process of NK cell, comprises the following steps:
Obtain CD34
+hemopoietic stem cell carries out stimulation in containing the stem cell media of hematopoietic stem cell growth factor and is divided into NK cell.
5. the external three-dimensional amplification cultivation method of NK cell according to claim 4, it is characterized in that, described hematopoietic stem cell growth factor comprises interleukin-2, IL-12, interleukin-15 and IL-18.
6. the external three-dimensional amplification cultivation method of NK cell according to claim 5, it is characterized in that, in described stem cell media, the concentration of interleukin-22 is the concentration that the concentration of 100-300U/ml, IL-12 is 0.5-4.5ng/ml, the concentration of interleukin-15 is 2-8ng/ml and IL-18 is 5-15ng/ml.
7. the external three-dimensional amplification cultivation method of NK cell according to claim 4, it is characterized in that, described hematopoietic stem cell growth factor comprises interleukin-2, and is also added with CD 3-resisting monoclonal antibody in described hematopoietic stem cell growth factor.
8. the external three-dimensional amplification cultivation method of NK cell according to claim 7, is characterized in that, in described stem cell media, the concentration of interleukin-2 is 5-15ng/ml, the concentration of CD 3-resisting monoclonal antibody is 5-15ng/ml.
9. the external three-dimensional amplification cultivation method of NK cell according to claim 4, it is characterized in that, the step of described NK cell being carried out to amplification in vitro cultivation is: ground to add in acetic acid by described chitin-sodium alginate and redissolve, and add the described stem cell media containing hematopoietic stem cell growth factor, inoculate described NK cell and carry out cultivation 20-50 days.
10. the external three-dimensional amplification cultivation method of NK cell according to claim 4, is characterized in that, described CD34
+the acquisition process of hemopoietic stem cell, comprises the following steps:
From hemocyte, isolate mononuclearcell, go out CD34 through magnetic bead sorting
+hemopoietic stem cell.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110195041A (en) * | 2019-06-20 | 2019-09-03 | 威海正生生物科技有限公司 | The method for obtaining high activity Tregs cell using ASC dimensional culture system |
| CN110951684A (en) * | 2019-12-20 | 2020-04-03 | 吉林省中科生物工程股份有限公司 | Human peripheral blood NK cell culture system and culture method |
| CN111925987A (en) * | 2020-09-24 | 2020-11-13 | 深圳市一五零生命科技有限公司 | NK cell serum-free culture solution and NK cell culture method |
| CN112409631A (en) * | 2020-12-02 | 2021-02-26 | 西北师范大学 | Preparation method and application of sodium alginate-chitosan porous composite material |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1628865A (en) * | 2003-12-17 | 2005-06-22 | 中国科学院大连化学物理研究所 | Method for preparing chitosan/sodium alginate tissue engineering stent |
| WO2008061392A1 (en) * | 2006-11-22 | 2008-05-29 | Hua Liu | Method for preparing cell populations with anti-tumor immune response activity |
| CN101773683A (en) * | 2010-03-03 | 2010-07-14 | 天津大学 | Chitosan modified alginate hydrogel three-dimensional porous bracket and preparation method thereof |
| CN102112600A (en) * | 2008-07-29 | 2011-06-29 | 株式会社绿十字 | Growth method for natural killer cells |
| CN102268405A (en) * | 2011-07-06 | 2011-12-07 | 安徽省立医院 | Method for auto NK (Natural Killer) cell in-vitro activation and amplification culture and special culture medium thereof |
| CN103923879A (en) * | 2014-04-29 | 2014-07-16 | 湖北华赛生物医药技术有限公司 | Preparation method and application of NK cytokine mixture |
| CN104711225A (en) * | 2015-04-09 | 2015-06-17 | 奥思达干细胞有限公司 | In-vitro preparation method of NK cells |
| CN104830774A (en) * | 2014-02-11 | 2015-08-12 | 天津卫凯生物工程有限公司 | Cell three-dimensional culture scaffold, preparation method and application thereof |
-
2015
- 2015-11-18 CN CN201510796650.6A patent/CN105441390A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1628865A (en) * | 2003-12-17 | 2005-06-22 | 中国科学院大连化学物理研究所 | Method for preparing chitosan/sodium alginate tissue engineering stent |
| WO2008061392A1 (en) * | 2006-11-22 | 2008-05-29 | Hua Liu | Method for preparing cell populations with anti-tumor immune response activity |
| CN102112600A (en) * | 2008-07-29 | 2011-06-29 | 株式会社绿十字 | Growth method for natural killer cells |
| CN101773683A (en) * | 2010-03-03 | 2010-07-14 | 天津大学 | Chitosan modified alginate hydrogel three-dimensional porous bracket and preparation method thereof |
| CN102268405A (en) * | 2011-07-06 | 2011-12-07 | 安徽省立医院 | Method for auto NK (Natural Killer) cell in-vitro activation and amplification culture and special culture medium thereof |
| CN104830774A (en) * | 2014-02-11 | 2015-08-12 | 天津卫凯生物工程有限公司 | Cell three-dimensional culture scaffold, preparation method and application thereof |
| CN103923879A (en) * | 2014-04-29 | 2014-07-16 | 湖北华赛生物医药技术有限公司 | Preparation method and application of NK cytokine mixture |
| CN104711225A (en) * | 2015-04-09 | 2015-06-17 | 奥思达干细胞有限公司 | In-vitro preparation method of NK cells |
Non-Patent Citations (1)
| Title |
|---|
| 李沁华 等: "冻干法制备的海藻酸钙-壳聚糖组织工程复合材料支架", 《中国组织工程研究》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110195041A (en) * | 2019-06-20 | 2019-09-03 | 威海正生生物科技有限公司 | The method for obtaining high activity Tregs cell using ASC dimensional culture system |
| CN110951684A (en) * | 2019-12-20 | 2020-04-03 | 吉林省中科生物工程股份有限公司 | Human peripheral blood NK cell culture system and culture method |
| CN111925987A (en) * | 2020-09-24 | 2020-11-13 | 深圳市一五零生命科技有限公司 | NK cell serum-free culture solution and NK cell culture method |
| CN112409631A (en) * | 2020-12-02 | 2021-02-26 | 西北师范大学 | Preparation method and application of sodium alginate-chitosan porous composite material |
| CN112409631B (en) * | 2020-12-02 | 2023-01-31 | 西北师范大学 | Preparation method and application of sodium alginate-chitosan porous composite material |
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