CN103146636A - Hiberarchy microcarrier and preparation method and application thereof - Google Patents

Abstract

The invention discloses a microcarrier with a hierarchical structure and its preparation method and application. The microcarrier with a hierarchical structure includes a primary structure and a secondary structure. The primary structure is a small droplet prepared by using a single emulsion microfluid. The size of the small droplet is 5-10 μm; the secondary structure is a large droplet generated by the internal phase of the double-emulsion microfluid, and the size of the large droplet is 200-500 μm; the preparation method includes firstly, the first stage of the microcarrier The preparation of the structure; secondly, the preparation of the secondary structure of the microcarrier; thirdly, the solidification of the droplet; the application of the microcarrier in the field of cell culture and multiple detection technology of protein, nucleic acid or cell.

Description

A kind of microcarrier with hierarchical structure and its preparation method and application

technical field

The invention relates to the field of biological analysis materials, in particular to a hierarchical structure microcarrier and its preparation method and application.

Background technique

Since Willhelm Roux first established in vitro cell culture by separating cells from chicken embryos in 1885, monolayer cell culture technology has a history of more than 100 years. The traditional monolayer cell culture technology refers to the in vitro cell culture method suitable for most anchorage-dependent cells, which need to be attached to a solid or semi-solid surface with an appropriate amount of positive charge for growth. It is mainly cultivated through some traditional culture bottles and multi-layer plates, but the operation is relatively cumbersome, the attachment area is limited, and the mass transfer and oxygen transfer are poor, which is greatly restricted in actual production and use.

In recent years, the research on three-dimensional cell culture in vitro has received widespread attention. Three-dimensional cell culture refers to the co-cultivation of carriers with different materials with three-dimensional structures and various types of cells in vitro, so that cells can grow in the three-dimensional structure of the carrier. Migrate and grow in the spatial structure to form a three-dimensional cell-carrier complex. Compared with traditional 2D-monolayer cell culture, drug, nutrient and gas diffusion properties in 3D cell culture are closer to living tissue. At present, three-dimensional cell culture has been widely used in basic biology and biomedical research. Its main advantage is that it has a good structure, which can directly reflect the relationship between structure and function. Neighboring cells form tighter connections.

Many natural materials have been used to make cell microcarriers and have been successfully commercialized. Most of the prior art utilizes spherical microcarriers to cultivate cells. Cells generally grow on the surface of microcarriers. When cells are cultured in large-scale agitation and suspension It is easy to be damaged by huge shear force.

Contents of the invention

The technical problem mainly solved by the present invention is to provide a hierarchical structure microcarrier, which can better maintain cell shape, reduce cost and improve culture efficiency.

In order to solve the above-mentioned technical problems, a technical solution adopted by the present invention is: provide a microcarrier with a hierarchical structure, the microcarrier with a hierarchical structure includes a primary structure and a secondary structure, and the primary structure is prepared by using a single emulsion microfluid The size of the small droplet is 5-10 μm; the secondary structure is a large droplet generated by the internal phase of the double-emulsion microfluid, and the size of the large droplet is 200-500 μm.

In a preferred embodiment of the present invention, the hierarchical microcarrier is an oil-soluble microcarrier, and the single-emulsion microfluidic device that generates the primary structure of the oil-soluble microcarrier is an oil-in-water (W/O) type , the double emulsion microfluidic device that generates the secondary structure of the oil-soluble microcarrier is a water-in-oil-in-water (W/O/W) type.

In a preferred embodiment of the present invention, the hierarchical microcarrier is a water-soluble microcarrier, and the single-emulsion microfluidic device forming the primary structure of the water-soluble microcarrier is a water-in-oil (O/W) type , the double emulsion microfluidic device forming the secondary structure of the water-soluble microcarrier is oil-in-water-in-oil (O/W/O).

In a preferred embodiment of the present invention, the hierarchical microcarrier is selected from polydimethylsiloxane or ethoxylated trimethylolpropane triacrylate.

In a preferred embodiment of the present invention, the hierarchical microcarrier is selected from collagen, chitosan, calcium alginate, agarose, polyhydroxyethyl methacrylate, polyethylene glycol diacrylate, acrylamide, One or more of N-isopropylacrylamide.

In a preferred embodiment of the present invention, the secondary structure of the hierarchical structure microcarrier is single capsule or multivesicle.

In order to solve the problems of the technologies described above, another technical solution adopted in the present invention is: provide a kind of method for preparing hierarchical microcarrier, comprising the following steps:

For the first time, the preparation of the primary structure of the microcarrier: According to the properties of the microcarrier, the pipeline of the microfluidic device is modified hydrophilically and hydrophobically, a suitable single-emulsion microfluidic device is assembled, and the dispersed phase and the continuous phase solution that are not compatible with each other are prepared to generate Emulsions containing droplets of small size;

Secondly, the preparation of the secondary structure of the microcarrier: Assemble a suitable double-emulsion microfluidic device, use the emulsion containing the primary structure as the intermediate phase solution, prepare the internal and external phase 1 solution that is incompatible with it, and adjust the phase solutions A flow rate of , generating liquid droplets that wrap several large-sized inner capsules and contain primary structures at the same time;

Thirdly, after solidifying the droplets and washing, the microcarriers with hierarchical structure can be obtained.

In a preferred embodiment of the present invention, the microfluidic device is selected from co-flow or converging microfluidic devices, and the pipe material of the microfluidic device is selected from silica, Teflon, polydimethyl One or more of siloxanes.

In a preferred embodiment of the present invention, the hydrophilic and hydrophobic modification is to modify the surface with hydroxyl or amino groups; the hydrophobic modification is to modify the surface with long-chain alkanes.

In order to solve the above technical problems, another technical solution adopted by the present invention is: the application of the hierarchical structure microcarrier in cell culture

In order to solve the above-mentioned technical problems, another technical solution adopted by the present invention is: the application of the hierarchically structured microcarriers in the technical field of multiple detection of proteins, nucleic acids or cells.

The beneficial effects of the present invention are: the present invention prepares the microcarrier into a porous structure, and the cells can enter the pores of the microcarrier to adhere and grow, which can protect the cells from the damage of stirring shear force and increase the surface area of cell adhesion , can better maintain the cell shape, reduce the cost, and improve the culture efficiency.

Description of drawings

Fig. 1 is the schematic diagram of hierarchical structure microcarrier of the present invention and preparation method;

Fig. 2 is that the present invention is the emulsion schematic diagram that prepares hierarchical structure microcarrier secondary structure;

Fig. 3 is a schematic diagram of a double emulsion microfluidic device of the present invention;

Fig. 4 is the microcarrier schematic diagram of hierarchical structure of the present invention;

The marks of each component in the accompanying drawings are as follows: 1. Outer phase; 2. Inner phase; 3. Intermediate phase; 5. Primary structure; 6. Secondary structure.

The preferred embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, so as to define the protection scope of the present invention more clearly.

Please refer to Figures 1-4, the embodiment of the present invention provides the following technical solutions

In one embodiment, a hierarchical structure microcarrier is provided, the hierarchical structure microcarrier includes a primary structure 5 and a secondary structure 6, the primary structure 5 is a small droplet prepared by utilizing single emulsion microfluidics, The size of the small droplet is 5-10 μm; the secondary structure 6 is a large droplet generated by the inner phase 2 of the double-emulsion microfluidics, and the size of the large droplet is 200-500 μm.

Preferably, the hierarchical structure microcarrier is an oil-soluble microcarrier, and the single-emulsion microfluidic device that generates the primary structure 5 of the oil-soluble microcarrier is an oil-in-water (W/O) type, and generates the oil-soluble microcarrier Microcarrier secondary structure 6 The double emulsion microfluidic device is of water-in-oil-in-water (W/O/W) type.

Preferably, the hierarchical microcarrier is a water-soluble microcarrier, and the single-emulsion microfluidic device forming the primary structure 5 of the water-soluble microcarrier is a water-in-oil (O/W) type, forming the water-soluble The secondary structure of the microcarrier 6 double emulsion microfluidic device is oil-in-water-in-oil (O/W/O).

Preferably, the microcarrier with hierarchical structure is selected from polydimethylsiloxane or ethoxylated trimethylolpropane triacrylate.

Preferably, the hierarchical microcarrier is selected from collagen, chitosan, calcium alginate, agarose, polyhydroxyethyl methacrylate, polyethylene glycol diacrylate, acrylamide, N-isopropylacrylamide one or more of.

Preferably, the secondary structure 6 of the hierarchically structured microcarrier is a single capsule or multiple capsules.

In another embodiment, a kind of method for preparing hierarchical microcarrier is provided, comprising the following steps:

First, the preparation of the primary structure 5 of the microcarrier: According to the properties of the microcarrier, modify the pipeline of the microfluidic device with hydrophilic and hydrophobic properties, assemble a suitable single-emulsion microfluidic device, and prepare a dispersed phase and a continuous phase solution that are immiscible with each other. Generate emulsions containing small-sized droplets;

Secondly, the preparation of microcarrier secondary structure 6: assemble a suitable double-emulsion microfluidic device, use the emulsion containing the primary structure as the intermediate phase 3 solution, prepare the internal and external phase 1 solution that is incompatible with it, and adjust each The flow rate of the phase solution generates droplets that wrap several large-sized inner capsules and contain primary structures at the same time.

Thirdly, after solidifying the droplets and washing, the microcarriers with hierarchical structure can be obtained. .

Preferably, the microfluidic device is selected from co-flow or converging microfluidic devices, and the pipe material of the microfluidic device is selected from one of silica, Teflon, and polydimethylsiloxane or more.

Preferably, the hydrophilic and hydrophobic modification is to modify the surface with hydroxyl or amino groups; the hydrophobic modification is to modify the surface with long-chain alkanes.

In another embodiment, the technical solution adopted in the present invention is that the application of the hierarchical microcarrier in cell culture

In another embodiment, the technical solution adopted in the present invention is the application of the hierarchically structured microcarrier in the technical field of multiple detection of proteins, nucleic acids or cells.

The various levels of structure of the hierarchical structure microcarriers are all prepared by microfluidics; the method for preparing the microcarriers with hierarchical structures by microfluidics is as follows: firstly, a single emulsion three-dimensional microfluidic device is used to generate a microfluidic device containing For the emulsion of small-sized monodisperse droplets, the primary structure 5 is the small-sized droplets, and then the emulsion containing small-sized droplets is used as the intermediate phase 3 solution of the double-emulsion microfluidic device, and the secondary structure 6 is the double-emulsion The large-sized droplets generated by the microfluidic inner phase 2 thus form microcarriers with a hierarchical structure after solidification.

The present invention firstly prepares the emulsion that contains many monodisperse small-size droplets through the single emulsion microfluid, and then uses this emulsion as the middle phase 3 solution of the double emulsion microfluid, through the shear action between the double emulsion and the flow rate of each phase solution control, forming microcarriers wrapped with several large-sized internal capsules (secondary structure 6), and these microcarriers also contain many small-sized liquid droplets as primary structures 5. Such a microcarrier with a hierarchical structure is a composite microcarrier that can meet the requirements of cell culture, protein, nucleic acid, and multiple detection of cells. The present invention designs and assembles a suitable single-emulsion microfluidic device to form many monodisperse small-sized droplets, and then this emulsion is used as the intermediate phase 3 solution of the double-emulsion microfluid. A droplet of small-sized endocysts, containing many small-sized endocysts. After solidification and cleaning, polymer microcarriers that meet the requirements of cell culture, protein, nucleic acid and cell detection can be formed.

The present invention's hierarchical structure microcarrier and its preparation method include designing and assembling a suitable single-emulsion microfluidic device to generate an emulsion containing many monodisperse small-sized droplets as the intermediate phase 3 solution of the double-emulsion microfluid, utilizing each phase The shear action between the solutions and the control of the flow rate of each phase solution generate droplets containing several large-sized inner capsules. These droplets also contain small-sized inner capsules, solidified droplets, and can be obtained after cleaning. Microcarriers, which can meet the requirements of cell culture and multiple detection of proteins, nucleic acids, cells, etc.

For hierarchical structure microcarriers and their preparation methods, the corresponding microfluid should be selected according to the properties of the microcarriers. For oil-soluble microcarriers, choose oil-in-water (W/O) single-emulsion microfluidics to form the primary structure 5, and choose water-in-oil-in-water (W/O/W) double-emulsion microfluidics to form the secondary structure 6. It can also be a water-soluble microcarrier, and the corresponding microfluidics should be selected. The microcarrier with hierarchical structure has certain biological applications. By adjusting the flow rate of each phase solution of the microfluidic preparation of primary structure 5 and secondary structure 6, it can be controlled to form a porous hierarchical structure with different sizes after solidification. Such hierarchical microcarriers can be used for cell culture and protein, nucleic acid and In the multivariate detection of cells. The porous structure of microcarriers with different sizes can adhere to cells or immobilize biomolecules for easy binding, reaction or further detection. When microcarriers with hierarchical structure are used in biological applications, cells can enter the interior of microcarriers through holes of different sizes. Large-sized holes can be used as scaffolds for cell adhesion, growth and proliferation, and small-sized holes can provide nutrients for cells. Substance and oxygen, thus, the microcarrier with hierarchical structure can become an excellent cell culture scaffold material.

The microcarrier with hierarchical structure and the preparation method thereof of the present invention, because it can accurately adjust the flow rate of each phase through microfluidics, and control the size and quantity of small-sized and large-sized inner capsules, can prepare microcarriers with a hierarchical structure, and After solidification, porous microcarriers with different sizes are formed. In biological applications, when the general ball carrier cultures cells, the cells usually grow on the surface of the microcarrier and grow in a single layer. The cells are easily damaged by the huge shear force during the suspension culture with stirring. When using microcarriers with a hierarchical structure to culture cells, cells can enter the interior of the microcarriers through the internal capsules of different sizes. Provide oxygen and nutrients, so that large-scale culture of adherent cells can be achieved in suspension, and the cells in the large-sized inner capsule have the opportunity to form aggregates. Therefore, this microcarrier with a hierarchical structure can become an excellent Cell culture scaffold material.

The microcarrier with hierarchical structure of the present invention is prepared by microfluidic technology, and its material itself has a wide range of choices. It can choose natural materials that are easy to obtain and have good biocompatibility, and can also choose artificial synthesis that is easy to synthesize and has strong controllability. Material. Due to the high controllability of microfluidic technology, microcarriers with hierarchical structure can be prepared, and after solidification, a penetrating porous structure of different sizes can be formed, which can be used as an excellent cell culture scaffold material. At the same time, the surface of this microcarrier can also be modified by some physical or chemical methods, which is not only better suitable for biological analysis, but also suitable for multiple detection of proteins, nucleic acids or cells.

Detailed ways

It should be understood that these examples are used to illustrate the present invention and not to limit the scope of the present invention. The unspecified implementation conditions are usually the conditions in routine experiments.

Example 1 Preparation of ethoxylated trimethylolpropane triacrylate (ETPTA) hierarchical structure microcarriers:

1. Preparation of the primary structure 5 of the ETPTA microcarrier: Select W/, single-emulsion glass capillary microfluidic device, and use octadecyltrimethoxysilane in 2%-10% acetone solution for hydrophobic treatment of the outer phase 1 glass capillary. Assemble the glass capillary microfluidic chip with glass capillary, glass slide, cover glass, sampling needle and quick-drying glue. The inner phase 2 is a 2wt% F108 solution, and the outer phase 1 is an ETPTA solution containing 1% photoinitiator. Connect the syringes filled with the biphasic solution to the corresponding glass capillary channels on the microfluidic chip. Adjust the two-phase flow rate to generate an ETPTA emulsion containing 5-10 μm small water droplets, collect and store.

2. Preparation of ETPTA microcarrier secondary structure 6: Select W/O/W type double-emulsion glass microfluidic device, treat mesophase 3 capillary with hydrophobic treatment, use 3-aminopropyltriethoxysilane (APTES) 2%-10% ethanol solution for hydrophilic treatment of the outer phase 1 capillary. The inner phase 2 is a 2wt% F108 solution, the middle phase 3 is an ETPTA emulsion containing many 5-10 μm water droplets, and the outer phase 1 is a mixed solution of 2wt% PVA and 2%wt F108 at a volume ratio of 1:1.

For the microfluidic device, wrap the chip and all the pipelines and syringes leading to the intermediate phase 3ETPTA with black tape or tin foil to prevent the device from being blocked during UV curing. Connect the syringes filled with the solutions of each phase to the corresponding glass capillary channels on the microfluidic chip. Adjust the three-phase flow rate until the microfluidics can stably generate ETPTA droplets that wrap several large-sized inner capsules and contain many small water droplets at the same time.

3. Preparation of microcarriers with hierarchical structure of ETPTA: Place a collection container at the end of the collection tube, put the outer phase 1 solution in it in advance, and immerse the end of the collection tube below the liquid surface. After the experimenter wears the UV protective equipment, turn on the UV spot curing The system light source illuminates the collection tube. The intensity and irradiation distance of the ultraviolet light can be adjusted according to the needs, so that the collected carrier can be completely cured. The collected ETPTA hierarchical structure microcarriers were washed with pure water and ethanol several times and then preserved.

Example 2 Preparation of microcarriers with hierarchical structure of polyethylene glycol diacrylate (PEGDA):

1. Preparation of PEGDA microcarrier primary structure 5: Select an O/W single-emulsion glass capillary microfluidic device, and assemble a glass capillary microfluidic chip with glass capillary, glass slide, cover glass, sampling needle and quick-drying adhesive. The inner phase 2 is a mixed solution of KF-96 (0.65CST) with lower viscosity and surfactant KF6015, and the outer phase 1 is a PEGDA hydrogel polymerization precursor solution containing 1% photoinitiator. Connect the syringes filled with the biphasic solution to the corresponding glass capillary channels on the microfluidic chip. Adjust the two-phase flow rate to generate a PEGDA emulsion containing 5-10 μm small oil droplets, collect and store.

2. Preparation of the secondary structure of PEGDA microcarriers: select O/W/O double emulsion glass microfluidic devices, and use 2%-10% ethanol solution of 3-aminopropyltriethoxysilane (APTES) for intermediate The capillary of phase 3 was treated to be hydrophilic, and the 2%-10% acetone solution of octadecyltrimethoxysilane was treated to be hydrophobic for the capillary of external phase 1. Inner phase 2 is a mixed solution of KF-96 (0.65CST) with lower viscosity and surfactant KF6015; middle phase 3 is a PEGDA hydrogel polymerization precursor solution containing 5-10 μm small oil droplets; outer phase 1 is a higher viscosity A mixed solution of high KF-96 (50CST) and surfactant KF6011.

For the microfluidic device, wrap the chip and all the pipelines and syringes leading to the mesophase 3PEGDA with black tape or tinfoil to prevent the device from clogging during UV curing. Connect the syringes filled with the solutions of each phase to the corresponding glass capillary channels on the microfluidic chip. Adjust the three-phase flow rate until the microfluidics can stably generate PEGDA droplets that wrap several large-sized inner capsules and contain many small oil droplets at the same time.

3. Preparation of microcarriers with PEGDA hierarchical structure: place a collection container at the end of the collection tube, put the outer phase 1 solution in it in advance, and immerse the end of the collection tube below the liquid surface, and turn on the UV spot curing after the experimenter puts on the UV protective equipment The system light source illuminates the collection tube. The intensity and irradiation distance of the ultraviolet light can be adjusted according to the needs, so that the collected carrier can be completely cured. The collected microcarriers with PEGDA hierarchical structure were washed with pure water and ethanol several times and then preserved.

Example 3 ETPTA hierarchical structure microcarriers are used for three-dimensional cell culture

1. Preparation of ETPTA hierarchical structure micro-carriers: Utilize O/W single-emulsion microfluidics to prepare ETPTA emulsions containing many small water droplets, and use this as the intermediate phase 3 solution of W/O/W double-emulsion microfluidics to prepare and wrap several Large water droplets, ETPTA hierarchical structure microcarriers containing many small water droplets at the same time, after curing, the microcarriers become penetrating porous hierarchical structures of different sizes.

2. Disinfection and sterilization of microcarriers with ETPTA hierarchical structure: Utilize an oxygen plasma treatment apparatus to make the surface of the microcarriers hydroxylated, then soak them in 75% alcohol solution overnight, then wash them repeatedly with sterile PBS (PH=7.4), and Soak in PBS and irradiate with ultraviolet light for 3h.

3. Cell inoculation: put the sterile microcarriers with ETPTA hierarchical structure in a six-well plate, inoculate the digested cells into the six-well plate, shake the six-well plate slightly for 3~4 hours, make the cells fully contact with the microcarriers, and inoculate them Place in a cell culture incubator (37°C, 5% CO2) and cultivate for 24-48h. It can also be directly placed into a microcarrier culture bottle for large-scale suspension culture.

4. Cell observation: After culture, use an optical microscope to observe whether the cells have adhered to the ETPTA hierarchical structure microcarrier holes and grown.

5. Detection of cell characterization and biological function: stain the cells, and use a fluorescence microscope to judge the survival rate of the cells; or use a scanning electron microscope to observe the shape and trend of cell growth. The kit is used to measure various biological indexes of the cells to evaluate the influence of the hierarchical structure microcarrier cultured cells on the biological functions of the cells.

The invention can better maintain the cell shape through the hierarchical structure microcarrier, reduce the cost and improve the culture efficiency.

The above is only an embodiment of the present invention, and does not limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made by using the description of the present invention and the contents of the accompanying drawings, or directly or indirectly used in other related technologies fields, all of which are equally included in the 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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