CN109306539B - 3D conductive cell culture scaffold and preparation method thereof - Google Patents

3D conductive cell culture scaffold and preparation method thereof Download PDF

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CN109306539B
CN109306539B CN201710627844.2A CN201710627844A CN109306539B CN 109306539 B CN109306539 B CN 109306539B CN 201710627844 A CN201710627844 A CN 201710627844A CN 109306539 B CN109306539 B CN 109306539B
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scaffold
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冯章启
史传梅
严珂
夏一鹭
李通
袁旭
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Nanjing University of Science and Technology
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Abstract

The invention discloses a 3D conductive cell culture scaffold and a preparation method thereof. The culture bracket is prepared by firstly mixing polyacrylonitrile and Fe3O4The high-dispersion polyacrylonitrile/Fe is prepared by electrostatic spinning technology and water phase device receiving3O4And (3) coating PEDOT on the surface of the fiber of the nano-fiber scaffold by in-situ polymerization, and loading graphene oxide on the outermost layer of the 3D scaffold by using electrostatic adsorption between the graphene oxide and the PEDOT. The average pore diameter among the fibers of the culture scaffold reaches 13.8 mu m, the culture scaffold has a three-dimensional porous structure with highly communicated inside and outside, and cells can smoothly migrate to the inside of the scaffold to form a uniform cell-3D culture system. Meanwhile, PEDOT with good conductivity and graphene oxide attract each other, and the graphene sheet layer is firmly attached to the outermost layer of the fiber. The culture scaffold has good biocompatibility and is beneficial to cell adhesion, growth and proliferation.

Description

3D conductive cell culture scaffold and preparation method thereof
Technical Field
The invention belongs to the technical field of biomedical materials, and relates to a 3D conductive cell culture support and a preparation method thereof.
Background
The three-dimensional cell culture Technique (TDCC) is a technique in which a carrier having a three-dimensional structure of different materials and various types of cells are co-cultured in vitro, and the cells are allowed to migrate and grow in the three-dimensional spatial structure of the carrier, thereby forming a three-dimensional cell carrier complex. Compared with the traditional two-dimensional plane culture, the three-dimensional culture mode is closer to the growth state of cells in the natural extracellular matrix and maintains the functional expression level of the cells, so that the in-vivo cell growth state can be simulated in vitro. Therefore, the three-dimensional culture technology can enable the result to be more accurate and reliable in the processes of drug screening and cytotoxicity detection.
Three-dimensional nanofiber scaffolds are widely used in the study of three-dimensional cell culture technology because they possess a structure similar to the natural extracellular matrix (Hogrebe N J, et al. biological chemistry: A patent regulator of biological cell biology and multicell organization [ J ]. Journal of biological Materials Research Part A, 2016.). The electrostatic spinning technology is used as an optimal preparation mode of the nanofiber and can be used for preparing a three-dimensional nanofiber scaffold. At present, the three-dimensional nanofiber scaffold based on the electrostatic spinning technology mainly has the following preparation strategies: adding a three-dimensional auxiliary receiving device; adding a pore-forming agent in the spinning process; superposing the electrostatic spinning nanofiber membrane; the self-assembly effect of the Polymer properties during spinning (Sun B, et al, Advances in the three-dimensional fibrous macro-crosslinking via electrospinning [ J ]. Progress in Polymer Science 2014,39(5): 862) 890). However, the pore size of the three-dimensional nanofiber scaffold prepared by the method is too small (<5 μm), cells cannot smoothly penetrate and migrate into the scaffold to form a uniform culture system, and therefore, the cells are still grown on the surface of the nanofiber scaffold in a manner similar to two-dimensional planar culture, and the existing state of the cells in a natural organism cannot be simulated.
The electrical stimulation cultured cells have very remarkable promotion effects on the aspects of directional differentiation of multifunctional stem cells, synapse elongation of nerve cells and the like, and the electrical stimulation is combined with a three-dimensional Cell culture scaffold, so that the cells can be endowed with a Growth environment similar to a natural extracellular matrix, and electrical stimulation is introduced at the same time, so that the electrical stimulation and the three-dimensional Cell culture scaffold jointly act on the cells to achieve a synergistic promotion effect (1.Tian H C, et al. Graphene Oxide connected polymer nano composite film for electrode-tissue interface. [ J ]. Biomaterials,2014,35(7): 2120. 2129.; 2.Chen C, et al. Biointerface by Cell Growth on Graphene Oxide bonded Bacterial Cell nanoparticles/Poly (3,4-ethylene dioxythioethylene) nanoparticles [ J. acids & 2016 (16): 358). The existing three-Dimensional conductive scaffold generally only has a macroscopic three-Dimensional structure, and the synergistic promotion effect of the two cannot be reflected when electrical stimulation induction is carried out (Chen C, et al, three-Dimensional BC/PEDOT Composite nanoparticles with High Performance for Electrode-Cell Interface [ J ]. Acs Applied Materials & Interfaces,2015,7(51): 28244.). It is therefore necessary to prepare a three-dimensional nanofiber scaffold with a suitable pore size for cell migration.
Disclosure of Invention
The invention aims to provide polyacrylonitrile/Fe with the pore diameter of more than 10 microns, good conductivity and biocompatibility3O4Polyethylene dioxythiophene/graphene oxide nano 3D conductive cell culture scaffold and a preparation method thereof.
The technical scheme for realizing the purpose of the invention is as follows:
A3D conductive cell culture scaffold is prepared from polyacrylonitrile and Fe3O4Highly discrete polyacrylonitrile/Fe is prepared by an electrostatic spinning technology and a specific receiving device3O4The preparation method comprises the following steps of preparing a nanofiber scaffold, wrapping polyethylene dioxythiophene (PEDOT) on the surface of scaffold fibers by in-situ polymerization, and loading graphene oxide on the outermost layer of the 3D scaffold by utilizing electrostatic adsorption between the graphene oxide and the PEDOT, wherein the preparation method specifically comprises the following steps:
step 1, Polyacrylonitrile/Fe3O4Preparing an electrostatic spinning solution:
mixing Fe3O4Placing the nano particles in N' N-dimethylformamide, uniformly dispersing by ultrasonic, adding Fe3O4Ultrasonically dispersing the Triton X-100 with equal mass again to be uniform, adding polyacrylonitrile protofilament, ultrasonically dissolving, and standing overnight to obtain polyacrylonitrile/Fe3O4Electrospinning solution of Fe3O4The concentration is 0.037-0.042 g/mL;
step 2, fluffy state polymerizationAcrylonitrile/Fe3O4Preparing the nano-fibers:
mixing polyacrylonitrile/Fe3O4Preparing the nanofiber by using the electrostatic spinning solution through a wet electrostatic spinning technology, taking a culture dish with a magnet at the bottom and with the water depth of 8-10 mm as a receiver, and carrying out vacuum freeze drying on the nanofiber to obtain fluffy polyacrylonitrile/Fe3O4A nanofiber;
step 3, fluffy polyacrylonitrile/Fe3O4Preparation of PEDOT:
adding polyacrylonitrile/Fe in fluffy state3O4Placing the nano-fiber in a mixed ether solution of ethylene dioxythiophene and ferric trichloride, carrying out ultrasonic closed reaction in an ice-water bath, repeatedly washing ethanol after the reaction is finished, replacing the ethanol in the bracket with water, and carrying out vacuum freeze drying to obtain fluffy polyacrylonitrile/Fe3O4PEDOT nanofibers;
step 4, preparing a graphene oxide dispersion liquid:
ultrasonically dispersing graphene oxide in water to obtain a graphene oxide dispersion liquid;
step 5, polyacrylonitrile/Fe3O4Preparing a PEDOT/graphene oxide 3D conductive scaffold:
adding polyacrylonitrile/Fe in fluffy state3O4Placing PEDOT nano-fiber in graphene oxide dispersion liquid, oscillating violently, standing overnight, washing with water to remove unloaded graphene sheet layer to obtain polyacrylonitrile/Fe3O4a/PEDOT/graphene oxide 3D conductive scaffold.
Preferably, in the step 1, the concentration of polyacrylonitrile is 0.125-0.15 g/mL, and Fe3O4The ultrasonic dispersion time is 0.8-1 h, the Triton X-100 ultrasonic dispersion time is 0.8-1 h, and the ultrasonic dispersion time of the polyacrylonitrile protofilament is 1.5-2 h.
Preferably, in the step 2, the magnet is a neodymium iron boron magnet, the spinning voltage is 15-16 kV, the flow rate of the spinning solution is 0.5-0.8 mL/h, and the receiving distance is 10-12 cm.
Preferably, in the step 3, the concentration of the ethylenedioxythiophene is 0.015-0.02 g/mL, and the concentration of the ferric chloride is 0.02-0.03 g/mL.
Preferably, in step 3, polyacrylonitrile/Fe in fluffy state3O4The nano-fiber is firstly dispersed in the ethylene dioxythiophene solution, and then the ferric chloride solution with the same volume as the ethylene dioxythiophene is added.
Preferably, in step 3, the water and ethanol replacement adopts gradient replacement, and the ratio of water to ethanol is 1:9, 3:7,5:5,7:3, and 9:1 in sequence.
Preferably, in the step 4, the concentration of the graphene oxide dispersion liquid is 0.5-1.5 mg/mL.
Compared with the prior art, the invention has the following advantages:
(1) fluffy polyacrylonitrile/Fe of the invention3O4The density of the nano-fiber is 0.7718mg/cm3The cell culture medium belongs to an ultra-light material, the average pore diameter among fibers reaches 13.8 mu m, the three-dimensional porous structure with highly communicated inside and outside is provided, and cells can smoothly migrate to the inside of a bracket to form a uniform cell-3D culture system;
(2) the positive charges on the surface of the PEDOT and the negative charges on the surface of the graphene oxide attract each other, so that the graphene sheet layer is firmly attached to the outermost layer of the fiber and is not easy to fall off, potential toxicity to cells is not caused, the 3D scaffold is endowed with good conductivity due to the introduction of the PEDOT, a supporting material is provided for the construction of an in-vitro model of cells in a natural organism in response to micro-current stimulation, and the graphene oxide loaded on the outermost layer of the scaffold has good biocompatibility and is beneficial to cell adhesion, growth and proliferation.
Drawings
FIG. 1 is a fluffy polyacrylonitrile/Fe3O4And (3) a real object diagram of the aqueous dispersion of the nano fibers.
FIG. 2 is a fluffy polyacrylonitrile/Fe3O4Scanning electron microscope images of the nanofibers.
FIG. 3 is polyacrylonitrile/Fe3O4Scanning electron microscope images of the/PEDOT/graphene oxide 3D conductive cell culture scaffold.
FIG. 4 is a fluffy polyacrylonitrile/Fe3O4Nanofiber, polypropylene-based/Fe3O4PEDOT nanofibers and polyacrylonitrile/Fe3O4Cyclic voltammetry characteristic curve of/PEDOT/graphene oxide 3D conductive cell culture scaffold.
FIG. 5 is a three-dimensional confocal scan of RGC-5 cultured on a 3D conductive scaffold.
FIG. 6 is the scanning electron microscope image of Hep G-2 cultured on 3D conductive scaffold.
FIG. 7 is a polyacrylonitrile/Fe prepared in comparative examples 1 and 23O4Scanning electron microscope images of the nanofibers.
Detailed Description
The present invention will be described in further detail with reference to the following examples and the accompanying drawings.
Example 1
(1) 3mL of N, N-dimethylformamide was weighed into a screw-top bottle, and 0.117g of Fe was weighed3O4Placing the nano particles in N, N-dimethylformamide, and performing ultrasonic dispersion for 1 h; weighing and mixing with Fe3O4Equal mass of Triton X-100 in said ultrasonically dispersed Fe3O4Continuing to perform ultrasonic dispersion for 1h in the dispersion liquid; 0.45g of polyacrylonitrile protofilament is weighed and added into the dispersion liquid, and the mixture is subjected to ultrasonic treatment for 1.5h and then is kept stand overnight to obtain the spinning liquid.
(2) A spinning device is set up, a culture dish with the depth of 8mm and the diameter of 22cm is used as a receiving device, and a neodymium iron boron magnet (10cm x 1cm) is placed at the bottom of the culture dish. Specific electrospinning parameters: 10cm 21# plain-head needle; the flow rate is 0.5 mL/h; the receiving distance is 12 cm; the voltage is 15 kV; the spinning time is 12 min. FIG. 1 is a diagram of a dispersed object of a nanofiber scaffold in water.
(3) Collecting the nano-fiber in the water bath in a centrifuge tube, adding two drops of ethanol into the centrifuge tube, oscillating uniformly, and freeze-drying for 60h to obtain fluffy polyacrylonitrile/Fe3O4And (3) nano fibers. FIG. 2 is a fluffy polyacrylonitrile/Fe3O4Scanning electron microscope image of the nano-fiber shows that the fiber is loaded with Fe from FIG. 23O4Nano-particles to make the surface rough, and Fe3O4Uniformly dispersed in the fiber without agglomeration; the pore diameter of the fiber scaffold is more than 10 mu m, and the pore diameter is favorable for smooth migration of cells to the scaffoldInside the frame, a uniform cell growth system is formed.
(4) Preparing 20mL of ether solution of ethylene dioxythiophene with the concentration of 0.017g/mL, placing the 3D nano-fiber scaffold prepared in the step (3) in the ether solution for oscillating and dispersing, preparing 20mL of ether solution of ferric trichloride with the concentration of 0.02g/mL, adding the ether solution of ethylene dioxythiophene, and carrying out ultrasonic reaction in an ice-water bath for 25 min. After the reaction is finished, the nano-fiber support is repeatedly washed by ethanol until ferric trichloride is completely removed, and then the nano-fiber support is gradient eluted by water-ethanol mixed solution (V)Ultrapure water：VEthanol1:9 respectively; 3: 7; 5: 5; 7: 3; 9:1), freeze-drying the eluted nano-fiber scaffold for 60 hours to obtain fluffy polypropylene/Fe3O4A PEDOT nanofiber conductive scaffold.
(5) Measuring 8mL of water into a screw bottle, and adding 8mg of graphene oxide into the screw bottle for ultrasonic dispersion for 2 hours to obtain a graphene oxide aqueous solution.
(6) And (5) placing the stent prepared in the step (4) in a graphene oxide aqueous solution, forcibly oscillating until the stent is uniformly dispersed in the graphene oxide aqueous solution, and standing overnight. Washing with water to remove the unadsorbed graphene oxide, and freeze-drying for 24h to obtain polyacrylonitrile/Fe3O4a/PEDOT/graphene oxide 3D conductive scaffold. Fig. 3 is a scanning electron micrograph of a 3D conductive scaffold, with the arrows indicating graphene oxide lamellae. The graph shows that the graphene sheet layer is tightly wrapped on the outer layer of the nanofiber, and no graphene oxide is filled in the micropores formed by crossing the nanofiber, so that the internal and external connectivity of the micropores of the 3D conductive support is not influenced, and the cells can be smoothly transferred to the inside of the support.
(7) Respectively adding polyacrylonitrile/Fe in a fluffy state3O4Nanofiber, polypropylene-based/Fe3O4the/PEDOT nano-fiber and the 3D conductive scaffold are dispersed in PBS buffer solution, and then used as a working electrode, a platinum electrode is used as a counter electrode, a calomel electrode is used as a reference electrode, the electrical property of the scaffold is detected in PBS (0.1M, PH is 7.4) by adopting cyclic voltammetry, and the scanning voltage: -0.4-0.6V; the sweep rate was 100mV/s, and the cyclic voltammetry characteristics tested are shown in FIG. 4. The ferroferric oxide nano particles have conductivity, soThe polyacrylonitrile/Fe3O4The nanofiber has a certain electrochemical performance, when PEDOT is loaded on the surface of the fiber through in-situ polymerization reaction, the conductivity of the nanofiber is increased, and the cyclic voltammetry current is obviously improved; when graphene oxide with poor conductor is loaded, the difference of cyclic voltammetry current is not large, and therefore good conductivity is still displayed.
Example 2
This example is essentially the same as example 1, the only difference being Fe3O4The doping amount is 0.1125g, the graphene oxide addition amount in the graphene oxide aqueous solution preparation is 4mg, and the performance of the prepared 3D conductive bracket is basically the same as that of the embodiment 1.
Example 3
The embodiment is basically the same as the embodiment 1, except that the concentration of the ethylenedioxythiophene is 0.02g/mL, the addition amount of the graphene oxide in the preparation of the graphene oxide aqueous solution is 12mg, and the performance of the prepared 3D conductive scaffold is basically the same as that of the embodiment 1.
Example 4
Three-dimensional culture of retinal ganglion cells
(1) The 3D conductive stent prepared in example 1 was soaked in 75% ethanol and sterilized for 8h, and then the conductive stent was washed 4 times with PBS to completely remove ethanol.
(2) Weighing 0.1g agarose powder, placing in 7mL PBS buffer solution, autoclaving, melting at 120 deg.C, spreading the agarose solution on the bottom of 48-well plate, cooling and solidifying the agarose, adding sterilized 3D conductive scaffold, adding 500 μ L DMEM complete culture medium containing 15% FBS, and placing the culture plate in CO2Incubate overnight in the incubator.
(3) Removing culture medium from 3D conductive scaffold, adding uniformly dispersed cell suspension of retinal ganglion cells (RGC-5) into 3D conductive scaffold, adding culture medium into culture plate to 500 μ L/well, placing in CO2Culturing in an incubator for 5 days. Cells were changed every 24 hours.
(4) Taking the 3D conductive bracket of the RGC-5 cells cultured for 5 days out of the spot plate, adding 20 mu L of Calcein-AM solution with the concentration of 2 mu mol/L, and dyeing for 15min in a dark place, and observing the shapes of the cells and the fibers under a three-dimensional confocal scanning microscope. The 3D scaffolds were supplemented with 20 μ L of medium every 15min of observation. Fig. 4 is a three-dimensional confocal scanning microscope image of the 3D conductive scaffold and the cells, from which it is apparent that the cells are tightly adhered to the 3D conductive scaffold, and the nanofibers are wrapped by the cell membrane proteins to expand and proliferate the cells along the direction of the nanofibers, thereby forming a wide cell-nanofiber composite network.
Three-dimensional culture of hepatoma cells
(1) The 3D conductive scaffold prepared in example 1 was soaked in 75% ethanol and sterilized for 8h, and then the conductive scaffold was washed 4 times with PBS to completely remove ethanol.
(2) Weighing 0.1g agarose powder, placing in 7mL PBS buffer solution, autoclaving, melting at 120 deg.C, spreading the agarose solution on the bottom of 24-well plate, cooling and solidifying the agarose, adding sterilized 3D conductive scaffold, adding 1mL DMEM containing 10% FBS, and placing the culture plate in CO2Incubate overnight in the incubator.
(3) Removing the culture medium in the 3D conductive bracket, adding uniformly dispersed liver cancer cell (HepG2) suspension into the 3D conductive bracket, wherein the cell amount is 2x105One/well, adding culture medium to 1 mL/well, placing in CO2Culturing in an incubator for 3 days. Cells were changed every 24 hours.
(4) After 3 days of cell culture, the culture medium in the culture plate was removed, the 3D conductive scaffold was washed 3 times with PBS buffer, 1ml of 2.5% glutaraldehyde in PBS was added to the conductive scaffold, and the conductive scaffold was sealed and left to stand at 4 ℃ for 4 hours in the dark to fix the cells.
(5) Removing glutaraldehyde solution from the conductive scaffold, washing with PBS 3 times, and gradient eluting with ethanol/water mixture (V)Ethanol:VWater (W)1:9,2:8,3:7,5:5,7:3,9:1,10: 0). And after freeze drying, spraying gold and observing the cell morphology under a scanning electron microscope. FIG. 6 is a scanning electron microscope image of Hep G2, in which cells are adhered to a 3D conductive scaffold and form obvious cell aggregates, which is beneficial to maintaining the in vitro culture functionalization of liver cancer cells, thereby being used for in vitro culture of liver cancerThe construction of the research model provides an ideal scaffold material.
Comparative example 1: Polyacrylonitrile/Fe3O4Preparation of nanofibers
(1) 3mL of N, N-dimethylformamide is weighed into a screw-top bottle, and 0.135g of Fe is weighed3O4Placing the nano particles in N, N-dimethylformamide, and performing ultrasonic dispersion for 1 h; weighing and mixing with Fe3O4Equal mass of Triton X-100 in said ultrasonically dispersed Fe3O4Continuing to perform ultrasonic dispersion for 1h in the dispersion liquid; 0.45g of polyacrylonitrile protofilament is weighed and added into the dispersion liquid, and the mixture is subjected to ultrasonic treatment for 1.5h and then is kept stand overnight to obtain the spinning liquid.
(2) A spinning device is set up, a culture dish with the depth of 8mm and the diameter of 22cm is used as a receiving device, and a neodymium iron boron magnet (10cm x 1cm) is placed at the bottom of the culture dish. Specific electrospinning parameters: 10cm 21# plain-head needle; the flow rate is 0.5 mL/h; the receiving distance is 12 cm; the voltage is 15 kV; the spinning time is 12 min. During spinning, due to Fe3O4Too large of an amount of incorporation causes the fibers to be rapidly attracted to the bottom of the receiver by the magnet, and thus the fibers are compressed by the influence of magnetic force when they are accumulated in water, FIG. 7a is a view showing the polyacrylonitrile/Fe prepared3O4The nano-fiber is scanned by an electron microscope, and as can be seen from the figure, the aperture between the fibers is mostly 5 mu m, the surface roughness of the fibers is obviously improved, and Fe appears3O4Agglomeration phenomenon, so the nanofiber is not suitable for being used as a support material for three-dimensional culture of cells.
Comparative example 2: Polyacrylonitrile/Fe3O4Preparation of nanofibers
(1) 3mL of N, N-dimethylformamide was weighed into a 5mL screw-top bottle, and 0.09g of Fe was weighed3O4Placing the nano particles in N, N-dimethylformamide, and performing ultrasonic dispersion for 1 h; weighing and mixing with Fe3O4Equal mass of Triton X-100 in said ultrasonically dispersed Fe3O4Continuing to perform ultrasonic dispersion for 1h in the dispersion liquid; 0.45g of polyacrylonitrile protofilament is weighed and added into the dispersion liquid, and the mixture is subjected to ultrasonic treatment for 1.5h and then is kept stand overnight to obtain the spinning liquid.
(2) Setting up a spinning device, and using a culture dish which is filled with ultrapure water with the depth of 8mm and has the diameter of 22cmAs a receiving device, a neodymium iron boron magnet (10cm by 1cm) was placed at the bottom of the culture dish. Specific electrospinning parameters: 10cm 21# plain-head needle; the flow rate is 0.5 mL/h; the receiving distance is 12 cm; the voltage is 15 kV; the spinning time is 12 min. During spinning, due to Fe3O4The doping amount is small, so that the magnetic force applied to the fibers cannot overcome the surface tension of water, and therefore, the fibers can only be accumulated on the water surface and cannot form a discrete nanofiber structure in the water. FIG. 7b is the polyacrylonitrile/Fe prepared3O4Scanning electron microscope image of nano fiber, wherein the fiber surface is smooth and has Fe3O4The fiber is distributed on the fiber, but the pore diameter among the fibers is mostly 5 mu m, and the fiber winding phenomenon occurs, so the fiber is not suitable to be used as a cell three-dimensional culture support material.

Claims (6)

1. A preparation method of a 3D conductive cell culture scaffold is characterized by comprising the following specific steps:
step 1, Polyacrylonitrile/Fe3O4Preparing an electrostatic spinning solution:
mixing 0.117g or 0.1125g Fe3O4Placing the nano particles in 3mL of N' N-dimethylformamide, ultrasonically dispersing the nano particles uniformly, and adding Fe3O4Ultrasonically dispersing evenly again for Triton X-100 with equal mass, adding 0.45g of polyacrylonitrile protofilament, ultrasonically dissolving, and standing overnight to obtain polyacrylonitrile/Fe3O4Electrostatic spinning solution;
step 2, fluffy polyacrylonitrile/Fe3O4Preparing the nano-fibers:
mixing polyacrylonitrile/Fe3O4Preparing the nanofiber by using the electrostatic spinning solution through a wet electrostatic spinning technology, taking a culture dish with a magnet at the bottom and with the water depth of 8-10 mm as a receiver, and carrying out vacuum freeze drying on the nanofiber to obtain fluffy polyacrylonitrile/Fe3O4The nanofiber comprises a magnet, a spinning voltage is 15-16 kV, the flow rate of a spinning solution is 0.5-0.8 mL/h, and the receiving distance is 10-12 cm;
step 3, fluffy polyacrylonitrile/Fe3O4Preparation of PEDOT:
adding polyacrylonitrile/Fe in fluffy state3O4Placing the nano-fiber in a mixed ether solution of ethylene dioxythiophene and ferric trichloride, carrying out ultrasonic closed reaction in an ice-water bath, repeatedly washing ethanol after the reaction is finished, replacing the ethanol in the bracket with water, and carrying out vacuum freeze drying to obtain fluffy polyacrylonitrile/Fe3O4PEDOT nanofibers;
step 4, preparing a graphene oxide dispersion liquid:
ultrasonically dispersing graphene oxide in water to obtain graphene oxide dispersion liquid with the concentration of 0.5-1.5 mg/mL;
step 5, polyacrylonitrile/Fe3O4Preparing a PEDOT/graphene oxide 3D conductive scaffold:
adding polyacrylonitrile/Fe in fluffy state3O4Placing PEDOT nano-fiber in graphene oxide dispersion liquid, oscillating violently, standing overnight, washing with water to remove unloaded graphene sheet layer to obtain polyacrylonitrile/Fe3O4a/PEDOT/graphene oxide 3D conductive scaffold.
2. The preparation method according to claim 1, wherein in the step 1, the concentration of polyacrylonitrile is 0.125-0.15 g/mL, and Fe3O4The ultrasonic dispersion time is 0.8-1 h, the Triton X-100 ultrasonic dispersion time is 0.8-1 h, and the ultrasonic dispersion time of the polyacrylonitrile protofilament is 1.5-2 h.
3. The preparation method according to claim 1, wherein in the step 3, the concentration of the ethylenedioxythiophene is 0.015 to 0.02g/mL, and the concentration of the ferric chloride is 0.02 to 0.03 g/mL.
4. The method according to claim 1, wherein in step 3, polyacrylonitrile/Fe in a fluffy state3O4The nano-fiber is firstly dispersed in the ethylene dioxythiophene solution, and then the ferric chloride solution with the same volume as the ethylene dioxythiophene is added.
5. The preparation method according to claim 1, wherein in the step 3, the water and ethanol are replaced by gradient replacement, and the ratio of water to ethanol is 1:9, 3:7,5:5,7:3 and 9:1 in sequence.
6. The 3D conductive cell culture scaffold prepared by the preparation method according to any one of claims 1 to 5.
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