CN103146636A - Hiberarchy microcarrier and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a hiberarchy microcarrier and a preparation method and application thereof. The hiberarchy microcarrier comprises a first-level structure and a second-level structure, wherein the first-level structure comprises small liquid drops which are prepared from single emulsion microfluid, and the size of each small liquid drop is 5-10 microns; and the second-level structure comprises large liquid drops which are generated through the internal phase of double emulsion microfluid, and the size of each large liquid drop is 200-500 micros. The preparation method comprises the following steps of: firstly, preparing the first-level structure of the microcarrier; secondly, preparing the second-level structure of the microcarrier; and finally, solidifying the liquid drops. The microcarrier is used in the technical field of cell culture and multi-element detection on proteins, nucleic acid or cells.
Description
Technical field
The present invention relates to the bioanalysis Material Field, particularly relate to a kind of hierarchy microcarrier and its preparation method and application.
Background technology
From Willhelm Roux in 1885 from the chicken embryo isolated cell set up first cell in vitro and cultivate, the monolayer cell culture technology history of existing over one hundred year.Traditional monolayer cell culture technology refers to the external cell culture method that is applicable to most of anchorage-dependent cells, and this class cell need to be attached to the upper growth of the solid of appropriate positive charge or semi-solid surface.It is mainly to cultivate by some traditional culturing bottles, multi-layer planar, but operation is more loaded down with trivial details, and area of attachment is limited, and mass transfer and biography oxygen are poor, are subject to great restriction in actual production is used.
In recent years, the research that external three-dimensional cell is cultivated has been subject to people and has paid close attention to widely, three-dimensional cell is cultivated and to be referred to and will have the carrier of three-dimensional structure differing materials and various different types of cell in external co-cultivation, make cell can move in the three-dimensional space structure of carrier, grow, consist of three-dimensional cell-carrier complexes.Compare with traditional two dimension-monolayer cell culture, the gentle bulk diffusion attribute of medicine, nutritive substance during three-dimensional cell is cultivated is closer to biological tissue.Cell three-dimensional is cultivated and has been widely used in fundamental biological knowledge and biomedical research at present, its major advantage is to have good structure, the relation of the direct reflect structure of energy and function, the form of cell and microenvironment be more near the state in body, and can set up with adjacent cell and contact more closely.
A lot of natural materialss have been used to make cell microcarrier and successfully commercialization, mostly utilize spherical microcarrier culturing cell in prior art, cell is generally to be grown on the surface of microcarrier, and cell is easy to be subject to huge shearing force when extensive stirring suspension is cultivated and breakage occurs.
Summary of the invention
The technical problem that the present invention mainly solves is to provide a kind of hierarchy microcarrier, and this carrier is hierarchy, can keep preferably cellular form, reduces costs, and can also improve culture efficiency.
For solving the problems of the technologies described above, the technical scheme that the present invention adopts is: a kind of hierarchy microcarrier is provided, described hierarchy microcarrier comprises primary structure and secondary structure, described primary structure is the small droplets that utilizes single emulsion microfluid to prepare, and described small drop sizes is 5-10 μ m; Described secondary structure is the large drop by generating mutually in two emulsion microfluids, and described large drop size is 200-500 μ m.
In a preferred embodiment of the present invention, described hierarchy microcarrier is the oil soluble microcarrier, the single emulsion micro fluidic device that generates the primary structure of described oil soluble microcarrier is oil-in-water (W/O) type, and the two emulsion micro fluidic devices that generate the secondary structure of described oil soluble microcarrier are water-in-oil-in-water (W/O/W) type.
In a preferred embodiment of the present invention, described hierarchy microcarrier is water-soluble microcarrier, the single emulsion micro fluidic device that forms the primary structure of described water-soluble microcarrier is water-in-oil (O/W) type, and the two emulsion micro fluidic devices that form the secondary structure of described water-soluble microcarrier are water-in-oil bag oil (O/W/O).
In a preferred embodiment of the present invention, described hierarchy microcarrier is selected from polydimethylsiloxane or ethoxylated trimethylolpropane triacrylate.
In a preferred embodiment of the present invention, described hierarchy microcarrier is selected from one or more in collagen, chitosan, alginate calcium, agarose, poly hydroxy ethyl acrylate, polyethyleneglycol diacrylate, acrylamide, NIPA.
In a preferred embodiment of the present invention, the secondary structure of described hierarchy microcarrier is single capsule or many capsules.
For solving the problems of the technologies described above, another technical solution used in the present invention is: a kind of method for preparing the hierarchy microcarrier is provided, comprises the following steps:
First, the preparation of microcarrier primary structure: according to microcarrier character, the micro fluidic device pipeline is carried out hydrophilic and hydrophobic modify, assemble suitable single emulsion micro fluidic device, prepare immiscible disperse phase and continuous phase solution, generate the emulsion that contains the small size drop;
Secondly, the preparation of microcarrier secondary structure: assemble suitable two emulsion micro fluidic devices, with the emulsion that comprises primary structure as middle phase solution, prepare immiscible with it interior foreign minister 1 solution, by regulating the flow velocity of each phase solution, generate the drop that wraps up some large size capsula internas and contain simultaneously primary structure;
Again, solidify drop, after cleaning, can obtain to have the microcarrier of hierarchy.
In a preferred embodiment of the present invention, described micro fluidic device is selected from coflow formula or convergence type micro fluidic device, and the pipeline material of described micro fluidic device is selected one or more in silicon-dioxide, Teflon, polydimethylsiloxane.
In a preferred embodiment of the present invention, described close and distant water is modified to hydroxyl or amino in finishing; Described hydrophobically modified be long chain alkane in finishing.
For solving the problems of the technologies described above, another technical solution used in the present invention is: the application of described hierarchy microcarrier in cell cultures
For solving the problems of the technologies described above, another technical solution used in the present invention is: the application of described hierarchy microcarrier in the multivariate detection technical field of protein, nucleic acid or cell.
The invention has the beneficial effects as follows: the present invention becomes vesicular structure with preparation of microcarriers; cell just can enter and adhere to growth in the microcarrier hole; the injury of namely can Cell protection avoiding stirring shearing force; can increase the surface-area of cell adhesion again; can keep preferably cellular form; reduce costs, can also improve culture efficiency.
Description of drawings
Fig. 1 is hierarchy microcarrier of the present invention and preparation method's schematic diagram;
Fig. 2 is that the present invention is the emulsion schematic diagram of preparation hierarchy microcarrier secondary structure;
Fig. 3 is the two emulsion microfluidic device schematic diagram of the present invention;
Fig. 4 is hierarchy microcarrier schematic diagram of the present invention;
In accompanying drawing, the mark of each parts is as follows: 1, foreign minister; 2, interior phase; 3, middle phase; 5, primary structure; 6, secondary structure.
Below in conjunction with accompanying drawing, preferred embodiment of the present invention is described in detail, thereby so that advantages and features of the invention can be easier to be it will be appreciated by those skilled in the art that, protection scope of the present invention is made more explicit defining.
See also Fig. 1-4, the embodiment of the present invention provides following technical scheme
In one embodiment, provide a kind of hierarchy microcarrier, described hierarchy microcarrier comprises primary structure 5 and secondary structure 6, the small droplets of described primary structure 5 for utilizing single emulsion microfluid to prepare, and described small drop sizes is 5-10 μ m; The large drops of described secondary structure 6 for generating by phase 2 in two emulsion microfluids, described large drop size is 200-500 μ m.
Preferably, described hierarchy microcarrier is the oil soluble microcarrier, the single emulsion micro fluidic device that generates the primary structure 5 of described oil soluble microcarrier is oil-in-water (W/O) type, and the two emulsion micro fluidic devices that generate the secondary structure 6 of described oil soluble microcarrier are water-in-oil-in-water (W/O/W) type.
Preferably, described hierarchy microcarrier is water-soluble microcarrier, the single emulsion micro fluidic device that forms the primary structure 5 of described water-soluble microcarrier is water-in-oil (O/W) type, and the two emulsion micro fluidic devices that form the secondary structure 6 of described water-soluble microcarrier are water-in-oil bag oil (O/W/O).
Preferably, described hierarchy microcarrier is selected from polydimethylsiloxane or ethoxylated trimethylolpropane triacrylate.
Preferably, described hierarchy microcarrier is selected from one or more in collagen, chitosan, alginate calcium, agarose, poly hydroxy ethyl acrylate, polyethyleneglycol diacrylate, acrylamide, NIPA.
Preferably, the secondary structure 6 of described hierarchy microcarrier is single capsule or many capsules.
In another embodiment, provide a kind of method for preparing the hierarchy microcarrier, comprise the following steps:
At first, the preparation of microcarrier primary structure 5: according to microcarrier character, the micro fluidic device pipeline is carried out hydrophilic and hydrophobic modify, assemble suitable single emulsion micro fluidic device, prepare immiscible disperse phase and continuous phase solution, generate the emulsion that contains the small size drop;
Secondly, the preparation of microcarrier secondary structure 6: assemble suitable two emulsion micro fluidic devices, with the emulsion that comprises primary structure as centre 3 solution mutually, prepare immiscible with it interior foreign minister 1 solution, by regulating the flow velocity of each phase solution, generate the drop that wraps up some large size capsula internas and contain simultaneously primary structure.
Again, solidify drop, after cleaning, can obtain to have the microcarrier of hierarchy.。
Preferably, described micro fluidic device is selected from coflow formula or convergence type micro fluidic device, and the pipeline material of described micro fluidic device is selected one or more in silicon-dioxide, Teflon, polydimethylsiloxane.
Preferably, described close and distant water is modified to hydroxyl or amino in finishing; Described hydrophobically modified be long chain alkane in finishing.
In another embodiment, the technical solution used in the present invention is, the application of described hierarchy microcarrier in cell cultures
In another embodiment, the technical solution used in the present invention is, the application of described hierarchy microcarrier in the multivariate detection technical field of protein, nucleic acid or cell.
Each level structure of described hierarchy microcarrier is all that the method by microfluid is prepared from; The method for preparing described microcarrier with hierarchy by microfluid is as follows: at first utilize the three-dimensional microflow control device of single emulsion to generate the emulsion that contains the single dispersant liquid drop of small size, primary structure 5 is the small size drop, then will contain the emulsion of small size drop as phase 3 solution in the middle of two emulsion micro fluidic devices, secondary structure 6 is the large size drops that in two emulsion microfluids, phase 2 generates, and forms the microcarrier with hierarchy after solidifying thus.
At first the present invention prepares by single emulsion microfluid and contains many single emulsions of disperseing the small size drop, then with middle mutually 3 solution of this emulsion as two emulsion microfluids, control by the shearing action between two emulsions and each phase solution flow rate, the microcarrier of several large size capsula internas (secondary structures 6) has been wrapped up in formation, also contains simultaneously many small size drops as primary structure 5 in these microcarriers.The microcarrier that has like this hierarchy is a kind ofly can meet cell cultures, protein, the compound microcarrier that nucleic acid and cell multivariate detection etc. require.Single emulsion micro fluidic device that design and assembly of the present invention are suitable, form many monodispersed small size drops, then this emulsion is as phase 3 solution in the middle of two emulsion microfluids, by regulating the flow velocity of each phase solution, generation comprises the drop of several large size capsula internas, contains simultaneously many small size capsula internas.After solidifying cleaning, can form the micro polymer carrier of requirements such as meeting cell cultures and protein, nucleic acid and cell detection.
hierarchy microcarrier of the present invention and preparation method thereof comprises single emulsion micro fluidic device that design and assembly are suitable, generation comprises many single emulsions of disperseing the small size drop, with these middle 3 solution mutually as two emulsion microfluids, utilize shearing action and each control of solution flow rate mutually between each phase solution, generation comprises the drop of several large size capsula internas, contain simultaneously the small size capsula interna in these drops, solidify drop, can obtain having the microcarrier of hierarchy after cleaning, this microcarrier can meet cell cultures and protein, nucleic acid, the requirement of the multivariate detection such as cell.
Hierarchy microcarrier and preparation method thereof should according to the character of microcarrier, be selected corresponding microfluid.Can be the oil soluble microcarrier, select the single emulsion microfluid of oil-in-water (W/O) to generate primary structure 5, select the two emulsion microfluids of water-in-oil-in-water (W/O/W) to form secondary structure 6.Also can be water-soluble microcarrier, should select the microfluid of respective opposite.This microcarrier with hierarchy has certain biological applications.Prepare each flow velocity of solution mutually of primary structure 5 and secondary structure 6 microfluids by adjusting, can control it and form afterwards the porous hierarchical structure that runs through not of uniform size in curing, such classification microcarrier can be used in the multivariate detection of cell cultures and protein, nucleic acid and cell.Microcarrier vesicular structure not of uniform size can adherent cell or fixing biological molecules, is convenient in conjunction with, reaction or further detects.Has the microcarrier of hierarchy when carrying out biological applications, cell can enter microcarrier inside by its hole not of uniform size, the large size hole can be used as the support of cell adhesion, growth and propagation, the small size hole can provide nutritive substance and oxygen for cell, thus, this microcarrier with hierarchy can become a kind of good cell culturing rack material.
Hierarchy microcarrier of the present invention and preparation method thereof, because it can regulate each phase flow velocity accurately by microfluid, control the size and number that generates small size and large size capsula interna, therefore can prepare the microcarrier with hierarchy, and form afterwards in curing and have the porous microcarrier that runs through not of uniform size.In biological applications, during general ball carrier culturing cell, cell normally is grown in the surface of microcarrier, is monolayer growth, and cell is subject to stirring suspension huge shearing force and breakage occurs when cultivating.And utilize when having the microcarrier culturing cell of hierarchy, cell can enter microcarrier inside by its capsula interna not of uniform size, the large size capsula interna can be used as the support of cell adhesion, growth and propagation, the small size capsula interna can provide oxygen and nutritive substance for cell, so can realize the suspension large scale culturing of attached cell, and cell has the chance that generates coacervate in the large size capsula interna, therefore, this microcarrier with hierarchy can become a kind of good cell culturing rack material.
Hierarchy microcarrier of the present invention is prepared from by micro-fluidic technologies, and the range of choice of its material own is wide, can select easy acquisition, natural materials that biocompatibility is good, also can select easily synthetic, synthetic material that controllability is strong.Due to the high controllability of micro-fluidic technologies, can prepare the microcarrier with hierarchy, form afterwards the vesicular structure that runs through not of uniform size in curing, can be used as a kind of good cell culturing rack material.Simultaneously, this microcarrier can also not only can better be applicable to bioanalysis by some physics or chemical process to its modifying surface, also is applicable to the fields such as multivariate detection of protein, nucleic acid or cell.
Embodiment
Should be understood that these embodiment are not limited to limit the scope of the invention for explanation the present invention.Not marked implementation condition is generally the condition in normal experiment.
The preparation of embodiment 1 ethoxylated trimethylolpropane triacrylate (ETPTA) hierarchy microcarrier:
1.ETPTA the preparation of microcarrier primary structure 5: select W/, single emulsion glass capillary micro fluidic device is made hydrophobic treatment with the 2%-10% acetone soln of octadecyl Trimethoxy silane to foreign minister's 1 glass capillary.Utilize glass capillary, slide glass, cover glass, point sample syringe needle and rapid dry glue assembling glass capillary micro-fluidic chip.Interior phase 2 is the F108 solution of 2wt%, and foreign minister 1 is for containing the ETPTA solution of 1% light trigger.The syringe that two phase liquid is housed is connected to corresponding glass capillary passage on micro-fluidic chip.Regulate the two-phase flow velocity, generate the ETPTA emulsion that contains the little water droplet of 5-10 μ m, Collection and conservation.
2.ETPTA the preparation of microcarrier secondary structure 6: two emulsion glass micro-fluidic devices of selecting the W/O/W type, to the centre mutually 3 kapillaries make hydrophobic treatment, carry out foreign minister's 1 kapillary is made hydrophilic treatment with the 2%-10% ethanolic soln of 3-aminopropyl triethoxysilane (APTES).Interior phase 2 is the F108 solution of 2wt%, and is middle mutually 3 for containing the ETPTA emulsion of the little water droplet of many 5-10 μ m, and foreign minister 1 is that 2wt%PVA and 2%wt F108 are with the mixing solutions of 1:1 volume ratio.
Micro fluidic device wraps up chip and all logical middle pipeline and the syringe of 3ETPTA mutually with black tape or tinfoil, when preventing ultra-violet curing, and blocking device.The syringe that each phase solution is housed is connected to corresponding glass capillary passage on micro-fluidic chip.Regulate the three-phase flow velocity, generate several large size capsula internas of parcel to the miniflow physical efficiency is stable, and contain simultaneously the ETPTA drop of many little water droplets.
3.ETPTA the preparation of hierarchy microcarrier: place collection container at the collection tube end, wherein put in advance foreign minister's 1 solution, and make below collection tube end immersed in liquid level, after the experimenter has dressed the uv-protection articles for use, open ultraviolet luminous point cure system light source, the irradiation collection tube.Can adjust as required ultraviolet light intensity and irradiation distance, so that the carrier of collecting solidifies fully.The ETPTA hierarchy microcarrier of collecting utilizes pure water, ethanol repeatedly to clean rear preservation.
The preparation of embodiment 2 polyethyleneglycol diacrylates (PEGDA) hierarchy microcarrier:
1.PEGDA the preparation of microcarrier primary structure 5: select the single emulsion glass capillary of O/W micro fluidic device, utilize glass capillary, slide glass, cover glass, point sample syringe needle and rapid dry glue assembling glass capillary micro-fluidic chip.Interior phase 2 is the mixing solutions of the lower KF-96 of viscosity (0.65CST) and tensio-active agent KF6015, and foreign minister 1 is for containing the PEGDA hydrogel polymerization precursor solution of 1% light trigger.The syringe that two phase liquid is housed is connected to corresponding glass capillary passage on micro-fluidic chip.Regulate the two-phase flow velocity, generate the PEGDA emulsion that contains the little oil droplet of 5-10 μ m, Collection and conservation.
2.PEGDA the preparation of microcarrier secondary structure: two emulsion glass micro-fluidic devices of selecting the O/W/O type, 2%-10% ethanolic soln with 3-aminopropyl triethoxysilane (APTES) carries out centre phase 3 kapillaries are made hydrophilic treatment, and the 2%-10% acetone soln of octadecyl Trimethoxy silane carries out hydrophobic treatment to foreign minister's 1 kapillary.Interior phase 2 is the mixing solutions of the lower KF-96 of viscosity (0.65CST) and tensio-active agent KF6015; Middle phase 3 is for containing the PEGDA hydrogel polymerization precursor solution of the little oil droplet of 5-10 μ m; Foreign minister 1 is the mixing solutions of the higher KF-96 of viscosity (50CST) and tensio-active agent KF6011.
Micro fluidic device wraps up chip and all logical middle pipeline and the syringe of 3PEGDA mutually with black tape or tinfoil, when preventing ultra-violet curing, and blocking device.The syringe that each phase solution is housed is connected to corresponding glass capillary passage on micro-fluidic chip.Regulate the three-phase flow velocity, generate several large size capsula internas of parcel to the miniflow physical efficiency is stable, and contain simultaneously the PEGDA drop of many little oil droplets.
3.PEGDA the preparation of hierarchy microcarrier: place collection container at the collection tube end, wherein put in advance foreign minister's 1 solution, and make below collection tube end immersed in liquid level, after the experimenter has dressed the uv-protection articles for use, open ultraviolet luminous point cure system light source, the irradiation collection tube.Can adjust as required ultraviolet light intensity and irradiation distance, so that the carrier of collecting solidifies fully.The PEGDA hierarchy microcarrier of collecting utilizes pure water, ethanol repeatedly to clean rear preservation.
Embodiment 3 ETPTA hierarchy microcarriers are used for three-dimensional cell and cultivate
1.ETPTA the preparation of hierarchy microcarrier: utilize the preparation of O/W type list emulsion microfluid to comprise the ETPTA emulsion of many little water droplets, and with these middle 3 solution mutually as the two emulsion microfluids of W/O/W type, several large water droplets of preparation parcel, the ETPTA hierarchy microcarrier that contains simultaneously many little water droplets, after solidifying, microcarrier becomes the porous hierarchical structure that runs through not of uniform size.
2.ETPTA the sterilization of hierarchy microcarrier: utilize the oxygen plasma treatment instrument to make the microcarrier surface hydroxylation, then it is soaked in 75% spirituous solution and spends the night, use again aseptic PBS (PH=7.4) repeatedly to clean, and be immersed in PBS medium ultraviolet irradiation 3h.
3. cell inoculation: aseptic ETPTA hierarchy microcarrier is placed in six orifice plates, and the cell that digestion is good is seeded in six orifice plates, slight wobble six orifice plate 3 ~ 4h, cell is fully contacted with microcarrier, be placed on cell culture incubator (37 ℃ 5%CO2), are cultivated 24-48h.Also can directly put into the microcarrier culturing bottle and carry out extensive suspension culture.
4. cell observation: after cultivating, utilize the observation by light microscope cell whether to adhere in ETPTA hierarchy microcarrier hole and grow.
5. the detection of cell sign and biological function: cell is dyeed, utilize the survival rate of fluorescent microscope judgement cell; Or utilize form and the trend of sem observation Growth of Cells.Utilize test kit that every biological indicator of cell is measured, to estimate this hierarchy microcarrier culturing cell to the impact of cell biological function.
The present invention can keep cellular form preferably by the hierarchy microcarrier, reduces costs, and can also improve culture efficiency.
The above is only embodiments of the invention; not thereby limit the scope of the claims of the present invention; every equivalent structure or equivalent flow process conversion that utilizes specification sheets of the present invention and accompanying drawing content to do; or directly or indirectly be used in other relevant technical fields, all in like manner be included in scope of patent protection of the present invention.
Claims (11)
1. a hierarchy microcarrier, is characterized in that, described hierarchy microcarrier comprises primary structure and secondary structure, and described primary structure is the small droplets that utilizes single emulsion microfluid to prepare, and described small drop sizes is 5-10 μ m; Described secondary structure is the large drop by generating mutually in two emulsion microfluids, and described large drop size is 200-500 μ m.
2. hierarchy microcarrier according to claim 1, it is characterized in that, described hierarchy microcarrier is the oil soluble microcarrier, the single emulsion micro fluidic device that generates the primary structure of described oil soluble microcarrier is oil-in-water (W/O) type, and the two emulsion micro fluidic devices that generate the secondary structure of described oil soluble microcarrier are water-in-oil-in-water (W/O/W) type.
3. hierarchy microcarrier according to claim 1, it is characterized in that, described hierarchy microcarrier is water-soluble microcarrier, the single emulsion micro fluidic device that forms the primary structure of described water-soluble microcarrier is water-in-oil (O/W) type, and the two emulsion micro fluidic devices that form the secondary structure of described water-soluble microcarrier are water-in-oil bag oil (O/W/O).
4. hierarchy microcarrier according to claim 2, is characterized in that, described hierarchy microcarrier is selected from polydimethylsiloxane or ethoxylated trimethylolpropane triacrylate.
5. hierarchy microcarrier according to claim 3, it is characterized in that, described hierarchy microcarrier is selected from one or more in collagen, chitosan, alginate calcium, agarose, poly hydroxy ethyl acrylate, polyethyleneglycol diacrylate, acrylamide, NIPA.
6. hierarchy microcarrier according to claim 1, is characterized in that, the secondary structure of described hierarchy microcarrier is single capsule or many capsules.
7. a method for preparing the arbitrary described hierarchy microcarrier of claim 1-6, is characterized in that, comprises the following steps:
At first, the preparation of microcarrier primary structure: according to microcarrier character, the micro fluidic device pipeline is carried out hydrophilic and hydrophobic modify, assemble suitable single emulsion micro fluidic device, prepare immiscible disperse phase and continuous phase solution, generate the emulsion that contains the small size drop;
Secondly, the preparation of microcarrier secondary structure: assemble suitable two emulsion micro fluidic devices, with the emulsion that comprises primary structure as middle phase solution, prepare immiscible with it interior foreign minister 1 solution, by regulating the flow velocity of each phase solution, generate the drop that wraps up some large size capsula internas and contain simultaneously primary structure;
Again, solidify drop, after cleaning, can obtain to have the microcarrier of hierarchy.
8. the method for preparation according to claim 7 hierarchy microcarrier claimed in claim 1, it is characterized in that, described micro fluidic device is selected from coflow formula or convergence type micro fluidic device, and the pipeline material of described micro fluidic device is selected one or more in silicon-dioxide, Teflon, polydimethylsiloxane.
9. the method for preparation according to claim 7 hierarchy microcarrier claimed in claim 1, is characterized in that, described close and distant water is modified to hydroxyl or amino in finishing; Described hydrophobically modified be long chain alkane in finishing.
10. the application of described hierarchy microcarrier as arbitrary in claim 1-6 in cell cultures.
11. the application of described hierarchy microcarrier as arbitrary in claim 1-6 in the multivariate detection technical field of protein, nucleic acid or cell.
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Application publication date: 20130612 |