CN115093703A - Polymer carrier with nano-pore structure for cell culture and preparation method and application thereof - Google Patents
Polymer carrier with nano-pore structure for cell culture and preparation method and application thereof Download PDFInfo
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- C08J9/00—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof
- C08J9/28—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof by elimination of a liquid phase from a macromolecular composition or article, e.g. drying of coagulum
- C08J9/286—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof by elimination of a liquid phase from a macromolecular composition or article, e.g. drying of coagulum the liquid phase being a solvent for the monomers but not for the resulting macromolecular composition, i.e. macroporous or macroreticular polymers
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0062—General methods for three-dimensional culture
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- C08J2201/0542—Precipitating the polymer by adding a non-solvent or a different solvent from an organic solvent-based polymer composition
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- C08J2201/054—Precipitating the polymer by adding a non-solvent or a different solvent
- C08J2201/0542—Precipitating the polymer by adding a non-solvent or a different solvent from an organic solvent-based polymer composition
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- C08J2381/00—Characterised by the use of macromolecular compounds obtained by reactions forming in the main chain of the macromolecule a linkage containing sulfur with or without nitrogen, oxygen, or carbon only; Polysulfones; Derivatives of such polymers
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Abstract
The invention discloses a polymer carrier with a nano-pore structure for cell culture and a preparation method and application thereof. The surface of the polymer carrier is in a nano-pore structure distribution, has good biocompatibility, can effectively promote the growth and proliferation of cells, realizes the full coverage of 2D to 3D culture of the cells, has low cost and simple process, and has good application prospect when being used as a culture cell substrate.
Description
Technical Field
The invention belongs to the technical field of biological materials for cell culture, and particularly relates to a polymer carrier with a nanopore structure for cell culture as well as a preparation method and application thereof.
Background
Stem cell therapy has great application prospect in the medical field, but the current cell expansion technology is difficult to meet the large use demand of stem cells. The hydrophilicity and the hydrophobicity and the appearance of the surface of a substrate for culturing cells can influence the mechanical transduction and the generation of intracellular force in the cells, and further influence the expression of genes, the growth, the migration and the proliferation of the cells. Therefore, the selection of suitable materials and morphologies to promote cell growth is the focus of current research.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a polymer carrier with a nanopore structure for cell culture, which has stable mechanical property and good biocompatibility, is beneficial to cell growth and improves cell culture efficiency, and a preparation method and application thereof.
In order to solve the technical problems, the invention adopts the following technical scheme.
A method for preparing a polymer carrier having a nanopore structure for cell culture, comprising the steps of:
(1) adding a polymer raw material into a good solvent, stirring and dissolving to obtain a polymer solution, wherein the polymer raw material is one or more of polyether sulfone, polysulfone, polyether ether ketone and polyether ketone, the mass fraction of the polymer solution is 12-30 wt%, and the good solvent comprises one or more of N, N-dimethylformamide, N-dimethylacetamide, dimethyl sulfoxide, N-methylpyrrolidone and concentrated sulfuric acid;
(2) and (2) dripping the polymer solution obtained in the step (1) into a poor solvent through an electrostatic ball-spraying process, wherein the poor solvent comprises alcohol, and performing phase separation to obtain the polymer microsphere serving as a polymer carrier with a nanopore structure for cell culture.
In the above method for preparing a polymer carrier with a nanopore structure for cell culture, preferably, the electrostatic microsphere process conditions are as follows: the positive voltage is controlled to be 2 kV-20 kV, the negative voltage is 0, the injection speed is 1 mm/min-10 mm/min, the vertical distance between the needle head and the liquid level is 20 cm-60 cm, the horizontal distance between the needle head and the liquid level (or the liquid drop incidence liquid level point) is 15 cm-40 cm, the needle head is a 90-degree glue dispensing needle head of 22G-30G, and the diameter of the obtained polymer microsphere is 0.2 mm-2 mm.
In the above method for preparing a polymer carrier with a nanopore structure for cell culture, preferably, the alcohol is 45-80% by volume.
In the above method for preparing a polymer carrier having a nanopore structure for cell culture, the polymer solution is preferably dropped under the condition of stirring the poor solvent at a stirring speed of 100rpm to 400 rpm.
The above method for preparing a polymer carrier having a nanopore structure for cell culture preferably further comprises removing a solvent, sterilizing, and washing after the phase separation.
In the above method for preparing a polymer carrier with a nanopore structure for cell culture, preferably, the diameter of the polymer microsphere is 0.2mm to 2mm, and small holes with a pore diameter of 1nm to 1000nm are uniformly distributed on the surface of the polymer microsphere.
As a general technical concept, the present invention also provides a method for preparing a polymer carrier having a nanopore structure for cell culture, comprising the steps of:
(1) adding a polymer raw material into a good solvent, stirring and dissolving to obtain a polymer solution, wherein the polymer raw material is one or more of polyether sulfone, polysulfone, polyether ether ketone and polyether ketone, the mass fraction of the polymer solution is 12-30 wt%, and the good solvent comprises one or more of N, N-dimethylformamide, N-dimethylacetamide, dimethyl sulfoxide, N-methylpyrrolidone and concentrated sulfuric acid;
(2) manually dropping the polymer solution obtained in the step (1) into a poor solvent in a stirring state, wherein the poor solvent comprises one or more of water, ethanol and methanol, and obtaining polymer microspheres as a polymer carrier with a nanopore structure for cell culture after phase separation.
In the above method for preparing a polymer carrier having a nanopore structure for cell culture, it is preferable that the manual dropping speed is 30 to 80 drops/min, the manual dropping is performed using a syringe, a needle of the syringe is a common injection needle having a diameter of 0.4 to 2.5mm, a vertical distance between the needle and a liquid surface is 20 to 60cm, and a stirring speed of a poor solvent is 100 to 400 rpm.
The above method for preparing a polymer carrier having a nanopore structure for cell culture preferably further comprises removing a solvent, sterilizing, and washing after the phase separation.
In the above method for preparing a polymer carrier with a nanopore structure for cell culture, preferably, the diameter of the polymer microsphere is 1mm to 3mm, and small holes with a pore diameter of 1nm to 1000nm are uniformly distributed on the surface of the polymer microsphere.
As a general technical concept, the present invention also provides a method for preparing a polymer carrier having a nanopore structure for cell culture, comprising the steps of:
(1) adding a polymer raw material into a good solvent, stirring and dissolving to obtain a polymer solution, wherein the polymer raw material is one or more of polyether sulfone, polysulfone, polyether ether ketone and polyether ketone, the mass fraction of the polymer solution is 12-30 wt%, and the good solvent comprises one or more of N, N-dimethylformamide, N-dimethylacetamide, dimethyl sulfoxide, N-methylpyrrolidone and concentrated sulfuric acid;
(2) and (2) coating the polymer solution obtained in the step (1) on a glass plate through spin coating, and then putting the glass plate into a poor solvent, wherein the poor solvent comprises one or more of water, ethanol and methanol, and after phase separation, obtaining a polymer film which is used as a polymer carrier with a nanopore structure for cell culture.
In the above method for preparing a polymer carrier with a nanopore structure for cell culture, preferably, the thickness of the polymer membrane is 1 μm to 2000 μm, and pores with a pore diameter of 1nm to 1000nm are uniformly distributed on the surface of the polymer membrane.
The above method for preparing a polymer carrier having a nanopore structure for cell culture preferably further comprises removing the solvent, sterilizing and washing after the phase separation.
As a general technical concept, the present invention also provides a polymer support with a nanopore structure for cell culture prepared by the above method for preparing a polymer support with a nanopore structure for cell culture.
As a general technical concept, the present invention also provides a use of the above polymer carrier having a nanopore structure for cell culture in cell culture.
Compared with the prior art, the invention has the advantages that:
(1) the nano-pores generated by phase separation are uniformly distributed on the surface of the polymer carrier membrane or microsphere prepared by the invention, and compared with non-porous and composite nano/micro-pore and micro-pore structures, the nano-pore structure on the polymer carrier prepared by the invention is beneficial to promoting cell adhesion, enhancing cell internal force and stimulating cell growth. Meanwhile, the nanopore structure is applied to the polymer 3D microsphere through phase separation, the transition from a polymer membrane to the microsphere realizes the full coverage of cell culture from two-dimensional (2D) to three-dimensional (3D), the specific surface area is improved, the three-dimensional cell culture is realized, and the cell amplification efficiency is promoted.
(2) The invention provides a method for preparing a polymer film or microsphere with a nano-pore structure by using a phase separation method, and experimental research proves that the nano-pore structure can effectively promote cell growth, and the polymer film and the microsphere can be used as good carriers for cell culture. The polymer microspheres of the 3D cultured cells prepared by the phase separation method are uniformly distributed with nanopores on the surface, have stable mechanical properties, high hardness and good biocompatibility, and are beneficial to cell growth and improvement of the cell culture efficiency. The preparation method comprises the following steps: the polymer is dissolved in a good solvent to obtain a uniform solution, and then the uniform solution is subjected to phase separation in a poor solvent by the method of the invention, so that the preparation of the carrier is realized.
(3) In the preparation method of the invention, the concentration of the polymer solution is also required, and if the concentration is too low, the microsphere shape is irregular, the concentration is too high, the microsphere surface is too compact, and a nano-pore structure cannot be formed. The control of the dropping speed of electrostatic spray balls or manual dropping balls is an important factor for ball forming, and if the speed is too high, the microspheres can be fused.
(4) The preparation method has low cost and simple and easy process; the selected polyether sulfone, polysulfone, polyether ether ketone or polyether ketone has good biocompatibility and mechanical property, and the membrane and microsphere carrier with good hydrophilicity and a nano-pore structure are favorable for cell adhesion, and have good application prospect when being used as a culture cell substrate.
Drawings
Fig. 1 is a schematic flow chart of a preparation method of a polyethersulfone membrane with a nanopore structure for cell culture in example 1 of the present invention.
Fig. 2 is a schematic flow chart of a preparation method of polyethersulfone microspheres with a nanopore structure for cell culture in example 2 and example 3 of the present invention.
Fig. 3 is a schematic process flow diagram of a preparation method of polyethersulfone membrane microspheres with a nanopore structure for cell culture in embodiment 4 of the invention.
Fig. 4 is a scanning electron microscope image of the nonporous polyethersulfone membrane prepared in investigation test 1, the nano polyethersulfone membrane prepared in example 1, the composite nano/micron polyethersulfone membrane prepared in comparative example 1, and the micron pore structure polyethersulfone membrane prepared in comparative example 2.
Fig. 5 is an enlarged scanning electron microscope image of the polyethersulfone membrane with a nano-pore structure prepared in example 1 of the invention.
FIG. 6 is a graph of water contact angles of plasma treated polystyrene (TCPS), Polyethersulfone (PES), polypropylene (PP), Polystyrene (PS), and Polydimethylsiloxane (PDMS) provided in investigation test 1 of the present invention.
FIG. 7 shows a 3h fluorescence microscope image of human adipose-derived stem cells (hASC) cultured on TCPS, PES, PP, PS, PDMS membranes according to investigation test 1 of the present invention.
FIG. 8 is a statistical chart of cell area of human adipose-derived stem cells (hASCs) 3h cultured on TCPS, PES, PP, PS, PDMS membranes according to investigation test 1 of the present invention.
FIG. 9 is a fluorescence microscope image of actin (F-actin) and cell nucleus (1), Paxillin (Paxillin) (2), myosin (pMyosin Iia) (3), vinculin (pFAK) (4), Lamin (Lamin A/C) (5) after culturing human adipose-derived stem cells (hASC) for 24h on the non-porous polyethersulfone membrane prepared in investigation test 1 of the present invention, the nano-polyethersulfone membrane prepared in example 1, the composite nano/micro polyethersulfone membrane of comparative example 1, and the micro-porous polyethersulfone membrane of comparative example 2, wherein the second and third rows are fluorescence microscope images of Paxillin (Paxillin) and the second row is an enlarged view of the square portion in the third row.
FIG. 10 is a statistical graph of fluorescence intensity of non-porous polyethersulfone membrane prepared in investigation test 1, nano polyethersulfone membrane prepared in example 1, composite nano/micro polyethersulfone membrane prepared in comparative example 1, and human adipose-derived stem cells (hASC) cultured on micro-pore structured polyethersulfone membrane prepared in comparative example 2 for 24h, cell area (1), Paxilin length (2), pMyosin IIa (3), pFAK (4), and Lamin A/C (5).
FIG. 11 is a scanning electron microscope image of polyethersulfone microspheres with a diameter of 500 μm and surface nanopore structures prepared in example 2 of the present invention.
FIG. 12 is a scanning electron microscope image of polyethersulfone microspheres with a diameter of 1mm and surface nanopore structures prepared in example 3 of the present invention.
FIG. 13 is a scanning electron microscope image of polyethersulfone microspheres with a diameter of 2mm and surface nanopore structures prepared in example 4 of the present invention.
FIG. 14 is a confocal laser microscopy image of actin (F-actin) cultured on 500 μm polyethersulfone microspheres prepared in example 2, of human adipose stem cells (hASC)35 d.
FIG. 15 is a confocal laser microscopy image of actin (F-actin) cultured in human adipose stem cells (hASC)35d on 1mm polyethersulfone microspheres prepared in example 3.
FIG. 16 is a confocal laser microscopy image of actin (F-actin) cultured on 2mm polyethersulfone microspheres prepared in example 4, of human adipose stem cells (hASC)35 d.
Detailed Description
The invention is further described below with reference to the drawings and specific preferred embodiments of the description, without thereby limiting the scope of protection of the invention. The materials and instruments used in the following examples are commercially available.
Example 1:
the invention relates to a preparation method of a polymer carrier with a nano-pore structure for cell culture, which adopts polyether sulfone as a raw material to prepare a polyether sulfone membrane, and comprises the following specific steps as shown in figure 1:
(1) weighing 12g of polyethersulfone, adding the polyethersulfone into 88g of good solvent DMAC (N, N-dimethylacetamide), stirring and dissolving, standing overnight, and removing air bubbles to obtain a polyethersulfone solution with the mass percentage of 12%.
(2) And (3) coating the obtained polyether sulfone solution on a glass plate through spin coating, putting the glass plate coated with the polyether sulfone solution into deionized water, and carrying out phase separation to obtain a polyether sulfone membrane, wherein the surface of the polyether sulfone membrane, which is in contact with water, is in a nano-pore structure, as shown in (2) of a figure and an enlarged view of the figure 5, and the surface of the polyether sulfone membrane, which is in contact with the glass plate, is in a composite nano/micro-pore structure.
(3) And (3) washing the obtained polyether sulfone membrane with hot water to remove the solvent, sterilizing with 75% alcohol by volume percentage, and cleaning with sterile PBS for 10 times to obtain the polyether sulfone membrane with the nano-pore structure.
The polyethersulfone membrane prepared according to the preparation method of the present example can be used as a carrier for 2D cultured cells.
Investigation test 1: the investigation of the polymer feedstock selection of this example comprises the following steps:
(1) weighing 12g of polyethersulfone, adding the polyethersulfone into 88g of good solvent DMAC (N, N-dimethylacetamide), stirring and dissolving, standing overnight, and removing air bubbles to obtain a polyethersulfone solution with the mass percentage of 12%.
(2) The obtained polyethersulfone solution was coated on a glass plate by spin coating, and placed in a vacuum chamber to evaporate the solvent and cause phase separation, to obtain a nonporous polyethersulfone membrane as shown in (1) of fig. 4.
(3) Non-porous plasma treated polystyrene membranes (TCPS), polypropylene membranes (PP), polystyrene membranes (PS) and polydimethylsiloxane membranes (PDMS) are purchased on the market, the water contact angles of the above membrane materials and the non-porous polyethersulfone membrane obtained in step (2) are tested under the same test conditions, and the polyethersulfone membrane (PES) has better hydrophilicity and is beneficial to protein adsorption by taking TCPS as reference, as shown in fig. 6, and comparing the water contact angles of the membrane materials.
(4) And (3) sterilizing the membrane material by adopting alcohol with the volume percentage of 75%, washing for 10 times by using sterile PBS, and then carrying out cell culture. Human adipose-derived stem cells (hASC) are cultured on TCPS, PES, PP, PS and PDMS membranes for 3h under the same test conditions, and observed by a fluorescence microscope, the cells spread on the membrane materials for 3h, as shown in FIG. 7, and the areas of the cells spread on the membrane materials are counted respectively, as shown in FIG. 8, as can be seen from FIGS. 7 and 8, based on TCPS, the cells spread on the PES membrane in the initial stage better, and the polyethersulfone used in the test can be used as a raw material to prepare a better carrier for culturing the cells, because the good hydrophilicity of the polyethersulfone is favorable for promoting the adhesion and growth of the cells, and the TCPS has good hydrophilicity, but cannot form spheres or has poor spheronization.
Investigation test 2: topography optimization test for this example
Cell culture under the same conditions was performed using four structures, non-porous, nanoporous, composite nano/microporous, and microporous polymer membranes, as shown in fig. 9 and 10, and it was found through observation with a fluorescence microscope that the cells adhered to the nanoporous membrane over a larger area and the intracellular force was greater, and thus the nanoporous structure stimulated cell growth. The nanopore region promotes integrin activation, enhances cell adhesion, and the micropores limit cytoskeletal assembly and intracellular force generation.
Comparative example 1:
the polyethersulfone membrane with the composite nano/micron pore structure is prepared by adopting polyethersulfone as a raw material, and the surface appearance is shown in figure 4 (3).
Comparative example 2:
the polyethersulfone membrane with the micron pore structure is prepared by adopting polyethersulfone as a raw material, and the surface appearance is shown in figure 4 (4).
Example 2:
the invention relates to a preparation method of a polymer carrier with a nanopore structure for cell culture, which adopts polyether sulfone as a raw material to prepare polymer microspheres, and comprises the following specific steps as shown in figure 2:
(1) weighing 12g of polyether sulfone, adding the polyether sulfone into 88g of good solvent DMAC (N, N-dimethylacetamide), stirring for dissolving, standing overnight, and removing bubbles to obtain a polyether sulfone solution with the mass percentage of 12%.
(2) Adding the obtained polyether sulfone solution into an injector, using a 90-degree needle, controlling a positive voltage of 9kV, a negative voltage of 0 and an injection speed of 4mm/min, stirring and dripping the solution into 75% alcohol by volume percentage, wherein the stirring speed is 200rpm, the vertical distance between the needle and the liquid surface is 40cm, the horizontal distance is 25cm, the needle is a 25G 90-degree dispensing needle, and phase separation is carried out to obtain polyether sulfone microspheres with the diameter of 500 mu m.
(3) Washing the obtained microsphere with hot water to remove solvent, sterilizing with 75% alcohol by volume, and washing with sterile PBS for 10 times to obtain polyethersulfone microsphere with nanopore structure, as shown in FIG. 11, with nanopores uniformly distributed on the surface of the microsphere.
The polymer microspheres prepared according to the embodiment can be used as carriers for 3D cultured cells, the surface nanopore structure stimulates the growth of the cells, and meanwhile, the 3D microspheres increase the specific surface area of materials and improve the cell amplification efficiency
The cells were cultured and observed by confocal laser microscopy to distribute and grow well on the surface of the microspheres, as shown in fig. 14.
Example 3:
a preparation method of a polymer carrier with a nanopore structure for cell culture adopts polyether sulfone as a raw material to prepare polymer microspheres, and comprises the following specific steps as shown in figure 2:
(1) weighing 12g of polyethersulfone, adding the polyethersulfone into 88g of good solvent DMAC (N, N-dimethylacetamide), stirring and dissolving, standing overnight, and removing air bubbles to obtain a polyethersulfone solution with the mass percentage of 12%.
(2) Adding the obtained polyether sulfone solution into an injector, using a 90-degree needle, controlling the positive voltage to be 6kV, the negative voltage to be 0 and the injection speed to be 2mm/min, stirring and dripping the solution into 75% alcohol by volume percentage, wherein the stirring speed is 200rpm, the vertical distance between the needle and the liquid surface is 40cm, the horizontal distance is 25cm, the needle is a 25G 90-degree dispensing needle, and performing phase separation to obtain polyether sulfone microspheres with the diameter of 1 mm.
(3) Washing the obtained microspheres with hot water to remove the solvent, sterilizing with 75% alcohol by volume percentage, and washing with sterile PBS for 10 times to obtain the polymer microspheres with a nanopore structure, wherein nanopores are uniformly distributed on the surfaces of the microspheres as shown in FIG. 12.
The polymer microspheres prepared according to the embodiment can be used as carriers for 3D cultured cells, the surface nanopore structure stimulates the growth of the cells, and meanwhile, the 3D microspheres increase the specific surface area of materials and improve the cell amplification efficiency
The cells were cultured, and the distribution of the cells on the surface of the microspheres and the growth thereof were observed by confocal laser microscopy, as shown in FIG. 15.
Example 4:
the preparation method of the polymer carrier with the nanopore structure for cell culture, disclosed by the invention, adopts polyether sulfone as a raw material to prepare the polymer microsphere, and comprises the following steps as shown in figure 3:
(1) weighing 12g of polyether sulfone, adding the polyether sulfone into 88g of good solvent DMAC (N, N-dimethylacetamide), stirring for dissolving, standing overnight, and removing bubbles to obtain a polyether sulfone solution with the mass percentage of 12%.
(2) Adding the obtained polyether sulfone solution into an injector, using a common needle with the diameter of 0.4mm, dripping into deionized water while stirring, wherein the stirring speed is 400rpm, the manual dripping speed is 60 drops/min, the vertical distance between the needle and the liquid surface is 50cm, and carrying out phase separation to obtain polyether sulfone microspheres with the diameter of 2 mm.
(3) Washing the obtained microspheres with hot water to remove the solvent, sterilizing with 75% alcohol by volume percentage, and washing with sterile PBS for 10 times to obtain the polymer microspheres with the nanopore structure, wherein nanopores are uniformly distributed on the surfaces of the microspheres as shown in FIG. 13.
The polymer microspheres prepared according to the embodiment can be used as a carrier for 3D cultured cells, the surface nanopore structure stimulates the growth of the cells, and meanwhile, the 3D microspheres increase the specific surface area of the material and improve the cell amplification efficiency. This process is simpler and easier to operate than example 3.
The cells were cultured and observed by confocal laser microscopy to distribute and grow well on the surface of the microspheres, as shown in fig. 16.
The polymer membrane or the polymer microsphere prepared by the invention can be used as a cell amplification or extracellular matrix extraction carrier, and the step of extracting the cells or the extracellular matrix comprises the steps of culturing the polyether sulfone microsphere loaded with the cells, enabling the cells to proliferate and secrete the extracellular matrix, then adding a good solvent to dissolve the polyether sulfone microsphere loaded with the cells and filtering to realize the extraction of the cells or the extracellular matrix (ECM).
The foregoing is merely a preferred embodiment of the invention and is not intended to limit the invention in any manner. Although the present invention has been described with reference to the preferred embodiments, it is not intended to be limited thereto. Those skilled in the art can make many variations and modifications to the disclosed embodiments, or equivalent variations, without departing from the spirit and scope of the invention, using the methods and techniques disclosed above. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention are still within the scope of the protection of the technical solution of the present invention.
Claims (10)
1. A method for preparing a polymer carrier with a nanopore structure for cell culture, comprising the following steps:
(1) adding a polymer raw material into a good solvent, stirring and dissolving to obtain a polymer solution, wherein the polymer raw material is one or more of polyether sulfone, polysulfone, polyether ether ketone and polyether ketone, the mass fraction of the polymer solution is 12-30 wt%, and the good solvent comprises one or more of N, N-dimethylformamide, N-dimethylacetamide, dimethyl sulfoxide, N-methylpyrrolidone and concentrated sulfuric acid;
(2) and (2) dripping the polymer solution obtained in the step (1) into a poor solvent through an electrostatic ball-spraying process, wherein the poor solvent comprises alcohol, and performing phase separation to obtain the polymer microsphere serving as a polymer carrier with a nanopore structure for cell culture.
2. The method for preparing a polymer carrier having a nanopore structure for cell culture according to claim 1, wherein the electrostatic bead process is performed under the following conditions: the positive voltage is controlled to be 2kV to 20kV, the negative voltage is 0, the injection speed is 1mm/min to 10mm/min, the vertical distance between the needle head and the liquid surface is 20cm to 60cm, the horizontal distance between the needle head and the liquid surface is 15cm to 40cm, the needle head is a 90-degree dispensing needle head of 22G to 30G, and the diameter of the obtained polymer microsphere is 0.2mm to 2 mm; the volume percentage content of the alcohol is 45-80 percent; dripping polymer solution under the condition of stirring poor solvent, wherein the stirring speed is 100 rpm-400 rpm; after the phase separation, the method also comprises the operations of removing the solvent, sterilizing and cleaning.
3. The method for preparing the polymer carrier with the nanopore structure for cell culture according to claim 1 or 2, wherein the diameter of the polymer microsphere is 0.2mm to 2mm, and the small holes with the pore diameter of 1nm to 1000nm are uniformly distributed on the surface of the polymer microsphere.
4. A method for preparing a polymer carrier with a nanopore structure for cell culture, which is characterized by comprising the following steps:
(1) adding a polymer raw material into a good solvent, stirring and dissolving to obtain a polymer solution, wherein the polymer raw material is one or more of polyether sulfone, polysulfone, polyether ether ketone and polyether ketone, the mass fraction of the polymer solution is 12-30 wt%, and the good solvent comprises one or more of N, N-dimethylformamide, N-dimethylacetamide, dimethyl sulfoxide, N-methylpyrrolidone and concentrated sulfuric acid;
(2) and (2) manually dripping the polymer solution obtained in the step (1) into a poor solvent in a stirring state, wherein the poor solvent comprises one or more of water, ethanol and methanol, and performing phase separation to obtain the polymer microspheres serving as a polymer carrier with a nanopore structure for cell culture.
5. The method for preparing a polymer carrier having a nanopore structure for cell culture according to claim 4, wherein the manual dropping speed is 30 to 80 drops/min, the manual dropping is performed using a syringe, a needle of the syringe is a general syringe needle having a diameter of 0.4 to 2.5mm, a vertical distance between the needle and a liquid surface is 20 to 60cm, and a stirring speed of the poor solvent is 100 to 400 rpm; after the phase separation, the method also comprises the operations of removing the solvent, sterilizing and cleaning.
6. The method for preparing the polymer carrier with the nanopore structure for cell culture according to claim 4 or 5, wherein the diameter of the polymer microsphere is 1mm to 3mm, and the small holes with the pore diameter of 1nm to 1000nm are uniformly distributed on the surface of the polymer microsphere.
7. A method for preparing a polymer carrier with a nanopore structure for cell culture, comprising the following steps:
(1) adding a polymer raw material into a good solvent, stirring and dissolving to obtain a polymer solution, wherein the polymer raw material is one or more of polyether sulfone, polysulfone, polyether ether ketone and polyether ketone, the mass fraction of the polymer solution is 12-30 wt%, and the good solvent comprises one or more of N, N-dimethylformamide, N-dimethylacetamide, dimethyl sulfoxide, N-methylpyrrolidone and concentrated sulfuric acid;
(2) and (2) coating the polymer solution obtained in the step (1) on a glass plate through spin coating, and then putting the glass plate into a poor solvent, wherein the poor solvent comprises one or more of water, ethanol and methanol, and after phase separation, obtaining a polymer membrane serving as a polymer carrier with a nanopore structure for cell culture.
8. The method for preparing the polymer carrier with the nanopore structure for cell culture according to claim 7, wherein the thickness of the polymer membrane is 1 μm to 2000 μm, and pores with the pore diameter of 1nm to 1000nm are uniformly distributed on the surface of the polymer membrane; after the phase separation, the method also comprises the operations of removing the solvent, sterilizing and cleaning.
9. A polymer carrier with a nanopore structure for cell culture prepared by the method for preparing the polymer carrier with a nanopore structure for cell culture according to any one of claims 1 to 8.
10. Use of the polymeric support for cell culture having a nanoporous structure according to claim 9 in cell culture.
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