JP2018196370A - Cell cultivation base material formed of multilayer film comprising conductive polymer layer and polymer layer for cell adhesion - Google Patents

Abstract

To provide a base material which serves as a foothold for efficiently producing a tissue, especially, a skeletal muscle or a nerve tissue in vitro.SOLUTION: The inventors have found a novel phenomenon in which, when producing a fiber foothold material having a laminated multilayer film or a coaxial two-layer structure in which a conductive polymer is a core and a biodegradable polymer is a sheath, instead of using a conductive polymer cell having a high conductance and an adhesion polymer and mixing them, a cell does not directly contact the conductive polymer in the fiber, however, on the fiber, a myotube formation efficiency is improved however no voltage is applied. There is provided a base material for cell cultivation formed into a fiber shape, the base material being a multilayer film base material formed of a conductive polymer layer and a cell adhesion layer, especially, the base material being a coaxial multilayer fiber in which, the conductive polymer is included therein and on an outermost layer, the cell adhesion polymer is coaxially laminated.SELECTED DRAWING: Figure 4

Description

  The present invention relates to a cell culture substrate composed of a multilayer film including a conductive polymer layer and a cell adhesion polymer layer, a cell differentiation induction and / or proliferation method using the substrate, and the like.

  For the purpose of application to cell engineering, regenerative medicine, etc., research has been conducted on a method for obtaining desired cells by promoting differentiation induction and / or proliferation of cultured cells, and a base material therefor.

  For example, for myoblasts that differentiate into myotubes and nerve cells that have nerve fibers or protrusion formation, these cells are efficiently cultured to promote differentiation induction and / or proliferation to produce desired cells. Establishment of technology is required. Since these cells have responsiveness to electrical signals, the conductive polymer is used as a culture substrate, and the conditions under which electrical stimulation is applied (Non-patent Document 1), utilization of the metal-containing conductive polymer Has been attempted (see Non-Patent Document 2).

  In order to efficiently produce skeletal muscle tissue in vitro, electrical stimulation is advantageous, and polypyrrole, polyaniline, etc. are known as conductive polymers (Patent Documents 1 and 2). Polyaniline is biodegradable and has cell adhesion properties. It has also been reported that myotube formation efficiency is improved without impressing voltage in the culture of myoblasts in a fibrous cell scaffold material mixed with a polymer having poly (procaprolactone) (Non-patent Document 3). ).

  Furthermore, the conductive polymer PPY / PLCL (polypyrrole / poly (l-lactic acid-co-ε-caprolactone)) is used as a scaffold for peripheral nerve cells. It has been reported that (axon) diameter enlargement promoting effect, nerve function recovery promoting effect, etc. are brought about (Non-patent Document 1).

JP 63-40211 A JP 2006-241613 A

Song J et al., Front Mol Neurosci. Vol. 9: Art. 117: 1-13 (2016). Yang H S et al., Adv Healthc Mater. 5: 137-145 (2016) S H Ku et al., Biomaterials, 33: 6098-6104 (2012).

  The present invention relates to a substrate serving as a scaffold for efficiently producing tissue, particularly skeletal muscle tissue or nerve tissue in vitro, a method for improving the production efficiency of the tissue, and a cell produced by the method It is an issue to provide.

  The present inventors use a conductive polymer layer and a cell adhesion polymer layer, which boast high conductivity, and do not mix them, but stack a cell adhesion polymer layer on the culture solution side of the conductive polymer layer. When a multi-layer fiber having a core / sheath structure or coaxial multilayer structure in which a cell adhesion polymer layer is laminated on the outer side of a multi-layer scaffold material, in particular, a conductive polymer layer is formed as a core, the cells are electrically The present invention was completed by finding a phenomenon that myotube formation efficiency was improved despite no direct contact with the polymer and no voltage applied to the fiber.

  Specifically, the present invention relates to a culture substrate for inducing and / or proliferating cells, and comprising a conductive polymer layer containing a conductive polymer and a cell adhesion polymer containing a cell adhesion polymer. A substrate that is a multilayer film having at least a two-layer structure including a layer is provided.

  In the substrate of the present invention, the cell is a cell having a fiber shape-forming ability, the multilayer film includes the fiber-like conductive polymer layer, and the outermost layer is coaxial with the conductive polymer layer, at least. There may be a multilayer fiber having a core / sheath structure or a coaxial multilayer structure by laminating one cell adhesion polymer layer.

  In the substrate of the present invention, the conductive polymer is selected from PEDOT / PSS [poly (3,4-ethylene.dioxythiophene) / poly (4-styrenesulfonic acid)], polyaniline or polypyrrole, and the cell adhesion Cell adhesion polymer containing polycaprolactone, polylactic acid, polyglycolic acid, or a copolymer thereof; polymer for general-purpose culture equipment containing polymethyl methacrylate or polystyrene; and biological origin containing collagen or gelatin The polymer may be at least one selected from the group consisting of:

  In the substrate of the present invention, the cells having the ability to form a fiber may be selected from myoblasts, nerve cells, established cell lines or primary cells, or model cells.

  The present invention also relates to a method for promoting cell differentiation induction and / or proliferation, which is based on a multilayer film including a conductive polymer layer containing a conductive polymer and a cell adhesion polymer layer containing a cell adhesion polymer. As a material, a method for culturing the cells is provided.

In the method of the present invention, the cell is a cell having a fiber shape-forming ability,
The base material is a coaxial multilayer fiber including a fiber-like conductive polymer layer and having at least one cell adhesion polymer layer as an outermost layer;
The cell may be cultured on the substrate.

In the method of the present invention, the conductive polymer is selected from PEDOT / PSS [poly (3,4-ethylene.dioxythiophene) / poly (4-styrenesulfonic acid)], polyaniline or polypyrrole,
The cell adhesion polymer is
A cell adhesive polymer comprising polycaprolactone, polylactic acid, polyglycolic acid or a copolymer thereof;
A polymer for general culture equipment comprising polymethyl methacrylate or polystyrene; and
A bio-derived polymer comprising collagen or gelatin;
And at least one selected from the group consisting of:

  In the method of the present invention, the cells having the ability to form a fiber may be selected from myoblasts, nerve cells, cell lines or primary cells thereof, or model cells thereof.

  Furthermore, the present invention provides a cell cultured using the substrate of the present invention.

  Furthermore, the present invention provides a cell that is cultured using the method of the present invention.

  In the cell of the present invention, the cell may be a tissue engineering cell or a regenerative medicine cell.

  According to the present invention, a substrate serving as a scaffold for efficiently producing tissue, particularly skeletal muscle tissue or nerve tissue in vitro, a method for improving the production efficiency of the tissue, and cells produced by the method The cells produced by these base materials and methods can be used in the fields of tissue engineering and regenerative medicine.

The figure which represents the electron microscope (SEM) image of the fiber scaffold about each material of PEDOT / PSS, polyaniline (polyaniline), polypyrrole (polypyrrole) (a scale bar represents 20 micrometers). Core sheath: a fiber scaffold having a core / sheath dual structure, blend liquid: a fiber scaffold when the core and sheath components are mixed, and sheath solution only: a fiber scaffold with only the sheath component. The figure showing the enlarged electron micrograph of the core and sheath fiber of FIG. Using PEDOT / PSS, sheath solution only: fiber scaffold formed with sheath solution only, core sheath: core / sheath structure double-layer fiber scaffold, blend solution: facilitating myotube formation in each fiber scaffold of core / sheath mixed solution Photograph showing effect (Scale bar represents 200 μm) The figure showing the result of having evaluated the myotube area occupation rate on the fiber scaffold preparation conditions using PEDOT / PSS. Sheath: Fiber scaffold formed only by sheath component (N = 4), Core sheath: Fiber scaffold with double core / sheath structure (N = 4), Blend: Fiber scaffold formed by mixing core / sheath component (N = 3), ** p <0.01, * p <0.05) The photomicrograph of the 0th day after changing to the differentiation induction culture solution in myotube formation evaluation in the case of changing the core flow rate is shown. The scale bar represents 200 μm, green represents the myosin heavy chain, and blue represents the nucleus. The photomicrograph of the 5th day after changing to the differentiation induction culture solution in the myotube formation evaluation in the case of changing the core flow rate is shown. The scale bar represents 200 μm, green represents the myosin heavy chain, and blue represents the nucleus. The figure showing the result of having evaluated myotube formation in the case of changing a core flow rate. (A) Change in cell density, (B) Occupied area ratio of cells expressing myosin heavy chain, (C) Fusion cell rate, (D) Myotube number, (E) Myotube length (Mean ± SD, *: p <0.05, **: p <0.01, n = 3). (A) Change in cell density during differentiation induction, (B) Occupied area ratio of cells expressing myosin heavy chain (MHC), (C) in each differentiation induction day when the concentration of sheath PCL / gelatin solution is changed Represents the number of myotubes, (D) myotube length, and (E) cell fusion rate (n = 1). The micrograph figure showing the change over time of myotube formation on the scaffold produced by changing the sheath PCL / gelatin solution concentration. Green represents myosin heavy chain (MHC), blue represents cell nucleus, and scale bar represents 200 μm.

1. One embodiment of the present invention is a culture substrate for inducing and / or proliferating cells, and comprising a conductive polymer layer containing a conductive polymer and a cell adhesion polymer. A base material that is a multilayer film having at least a two-layer structure including a polymer layer for cell adhesion. Preferably, the base material is a cell in which the cells have a fiber shape-forming ability, the multilayer film encloses the fiber-like conductive polymer layer, and the outermost layer is substantially coaxial with the conductive polymer layer. A multilayer fiber having a core / sheath structure or a substantially coaxial multilayer structure by laminating at least one cell adhesion polymer layer.

  In the substrate, the conductive polymer is selected from PEDOT / PSS [poly (3,4-ethylene.dioxythiophene) / poly (4-styrenesulfonic acid)], polyaniline or polypyrrole, Examples of adhesion polymers include cell adhesion polymers comprising polycaprolactone, polylactic acid, polyglycolic acid, or copolymers thereof; polymers for general culture equipment comprising polymethyl methacrylate or polystyrene; and collagen or gelatin A bio-derived polymer comprising: at least one selected from the group consisting of:

  A more specific example of the substrate of the present invention is when PEDOT / PSS is used as the conductive polymer. PEDOT / PSS is poly (3,4-ethylene dioxythiophene) doped with poly (4-styrene sulfonic acid), PEDOT has good conductivity, in doped (conductor) state It has excellent environmental stability and light transparency when used as a thin film. PEDOT coating can generally be performed using an aqueous dispersion consisting of a mixture of PEDOT and polyanionic poly (styrene sulfonate) or PEDOT-PSS.

  In the present specification, the “polymer for cell adhesion” refers to a multilayer polymer substrate of the present invention, which is disposed between a conductive polymer layer that does not directly contact cells and the cultured cells, and It refers to the polymer material of the polymer layer that is in direct contact. That is, the “cell adhesion polymer” is not limited to a polymer having an excellent cell adhesion activity, including a molecule having an activity of promoting cell adhesion, as long as cells adhere. In this specification, the “cell adhesion polymer” is classified and described as “cell adhesion polymer”, “polymer for general-purpose culture equipment”, and “bio-derived polymer” depending on the type of the material. However, these do not necessarily mean that they have excellent cell adhesion.

  Examples of cell adhesion polymers include cell adhesion polymers comprising polycaprolactone, polylactic acid, polyglycolic acid, or copolymers thereof; polymers for general culture equipment comprising polymethyl methacrylate or polystyrene; and collagen or Examples thereof include bio-derived polymers containing gelatin.

  In this specification, spin coating method, dipping method, spray coating method, electric field polymerization method, vapor deposition method, vapor deposition polymerization method, brush are used as the coating method for producing the laminated film constituting the multilayer polymer substrate. Known methods such as a coating method, a blade coating method, a roller coating method, and a roll-to-roll method can be used. Further, when producing a multilayer fiber having a core / sheath structure or a coaxial multilayer structure, an electrospinning method can be used.

  When the coaxial multilayer fiber is produced by electrospinning, a conductive polymer solution is used as a core solution, and a cell adhesion polymer solution or a raw material solution before the cross-linking reaction of the polymer is used as a sheath solution. A fiber having a coaxial two-layer structure that forms a core / sheath structure can be manufactured by laminating a biodegradable polymer on the outside of a conductive polymer as a core using a spinning device.

  A multilayer base material of a conductive polymer layer and a cell adhesion polymer layer produced by the above method or a coaxial two-layer fiber base material having these polymer layers is placed in a culture solution as a scaffold. By culturing the cells in an appropriate culture medium known to those skilled in the art, cell differentiation induction and / or cell proliferation can be promoted, and a desired tissue, tissue model, bioactuator and the like can be obtained. When a coaxial multilayer structure fiber is used as a base material, an artificial tissue in which cells are arranged along this scaffold base material can be obtained.

  In addition, the cells having the fiber shape-forming ability are included as long as they are cells capable of stretching cell fibers and cell processes, and more specific examples include cells and / or processes having the ability to form myotubes. Examples include cells that have the ability to stretch. Specifically, it is selected from myoblasts, nerve cells, their established cell lines, primary cells, or model cells. Examples of model cells include C2C12 cells as a myoblast model and PC12 cells as a neuronal cell model. In addition, cells prepared by inducing differentiation of induced pluripotent stem cells (iPS cells) or embryonic stem cells (ES cells) can be used.

  The cells used in the practice of the present invention may be cells derived from mammals, for example, cells derived from humans, mice, rats, guinea pigs, dogs, cats, monkeys, pigs, cows, horses, sheep or rabbits. Can be mentioned.

  When myoblasts are cultured with the scaffold, differentiation can be induced into myotubes. In addition, the induced pluripotent stem cell or embryonic stem cell is induced to differentiate into each of the cells, and then cultured together with the scaffold base material of the present invention, whereby cells having a desired fiber shape are obtained. Compared to the case where no is used, it can also be obtained in a short culture period.

  The cells produced using the culture substrate of the present invention can be applied to fields such as tissue engineering and regenerative medicine.

  When cells produced using the culture substrate of the present invention are used for tissue engineering, for example, in the field of drug discovery research, a three-dimensionally constructed cultured tissue is used as a tissue model for the effectiveness and safety of drugs. It can be used for research materials for evaluation and bioactuators as artificial muscles.

  In addition, when cells produced using the culture substrate of the present invention are used for regenerative medicine, for example, the cultured cells produced using the above-mentioned substrate are transplanted to a damaged site of a patient whose tissue has been damaged. By using it, it can be used as a material for regenerative medicine for regenerating damaged tissue. In particular, cells induced or proliferated using a coaxial multilayer fiber substrate can be used as cells for transplantation. Moreover, the repair of a damaged site can be accelerated | stimulated by transplanting the base material of this invention to a patient's damaged site.

  Diseases associated with tissue damage include tissues or organs affected by genetic defects, damage, lack of proper cell differentiation (eg, in cell proliferative disorders), normal body wear, or lifestyle. These include diseases such as lifestyle-related diseases that gradually deteriorate in function or structure over time. More specifically, examples of diseases related to tissue damage include, for example, traumatic tissue damage to brain tissue such as muscle tissue and brain contusion due to accidents such as traffic accidents, Alzheimer's disease, Parkinson's disease Tissue damage in neurodegenerative diseases such as Huntington's disease, multiple sclerosis and amyotrophic lateral sclerosis (ALS); for example, transverse myelitis, demyelination that occurs after trauma to the brain or spinal cord, acute brain injury Head injury, spinal cord injury, peripheral nerve injury, ischemic brain injury, hereditary myelin disorder of the CNS, epilepsy, perinatal asphyxia, asphyxia, hypoxia, status epilepticus, Shy-Drager syndrome, autism, and Tissue damage due to nervous system disorders such as stroke; for example, cancer such as lung cancer, liver cancer, or tissue damage caused by cytotoxicity of drugs resulting from anticancer treatment with chemotherapeutic agents; Or tissue damage due to metabolic disorders such as diabetes and Niemann-Pick disease; Tissue damage due to inflammation-related disorders; for example, eye damage such as glaucoma, retinitis pigmentosa, Norie disease, and macular degeneration; for example, circulation such as atherosclerosis, heart failure, myocardial infarction and angina Tissue damage due to genital disorders; for example, blood disorders such as Wiscott-Aldrich syndrome; muscular dystrophy; tissue damage due to digestive, renal, liver or lung diseases; for example, tissue damage due to adrenal diseases such as Addison's disease; For example, tissue caused by infectious tissue damage caused by infection with viruses or microorganisms such as hepatitis C infection and acquired immunodeficiency Scratches and the like.

2. Method for culturing cells having fiber shape forming ability Another embodiment of the present invention is a method for promoting the growth of cultured cells, comprising a conductive polymer layer containing a conductive polymer and a cell adhesion containing a cell adhesion polymer. The cell is cultured using a multilayer film including a polymer layer for use as a base material. In particular, in this method, the cell is a cell having a fiber shape-forming ability, and the base material includes a fiber-like conductive polymer layer, and the outermost layer has at least one cell adhesion polymer layer. A method of culturing the cells on the substrate, which is a substrate composed of multilayer fibers, is preferable.

  The conductive polymer and the cell adhesion polymer constituting the substrate used in the method for culturing fibrous cells of the present invention are prepared by using the materials described in the embodiment of the cell culture substrate and using the materials described above. A material can be produced and used in the method of the present invention.

  The cells cultured to form cells having a fiber shape are cells having the ability to form fiber shapes, and more specifically, cells and / or processes having the ability to form myotubes are stretched. For example, myoblasts are cells having the ability to form myotubes, and neurons are examples of cells having the ability to extend processes. Furthermore, these cell lines or primary cells, or these model cells can also be used. Examples of the model cells include C12C12 cells as myoblast models and PC12 cells which are model cells of nerve cells. In addition, induced pluripotent stem cells and embryonic stem cells can be induced to differentiate into these cells, and these cells can be used.

  Fibrous cells produced by the method for culturing fibrous cells are used for cell engineering, more specifically, tissue models or bioactuators for evaluating the effectiveness and safety of drugs in the field of drug discovery research. Or in the field of regenerative medicine, it can be used by transplanting the damaged site in order to promote regeneration of the tissue of a patient who has suffered tissue damage in the above-mentioned various diseases.

3. Cell having fiber shape Another embodiment of the present invention is a cell that has been induced and / or proliferated using the substrate and the culture method. In particular, the cells cultured using the multilayer fiber substrate or by the method using the multilayer fiber substrate are preferably cells having fiber shape-forming ability. Examples of cells having the ability to form myotubes include cells that have the ability to form myotubes and / or cells that have the ability to extend processes. A cell having an ability to form a myoblast and a cell having an ability to extend an axon from a neurite, that is, a nerve fiber, includes a nerve cell. Moreover, the cell line of these cells, a primary cell, or these model cells are mentioned.

  For example, by culturing these cells with a coaxial multilayer fiber substrate, the cells adhere to the substrate, and differentiation induction and / or proliferation is promoted compared to when the substrate is not used. Thereby, the cell which has a desired fiber shape is acquirable. In addition, tissues and tissue models composed of these cells can be used for tissue engineering, regenerative medicine, and the like.

  For example, when these cells are used for tissue engineering, more specifically, they can be used for a tissue model or a bioactuator for evaluating the effectiveness and safety of a drug in the field of drug discovery research.

  In addition, by transplanting cells having this fiber shape into an injured site of a patient with tissue damage, cells for regenerative medicine or tissues composed of these cells can be used as tissues for regenerative medicine. Examples of the target disease using cells or tissues for regenerative medicine include the various diseases described in the embodiment of the cultured cell substrate.

  In addition, by transplanting cells obtained by culturing myoblasts using the coaxial multilayer fiber base material to a damaged site of a patient having a muscle injury site, it is possible to shorten the period until healing for muscle injury, It can be used as a cell for regenerative medicine. In addition, by transplanting cells obtained by culturing nerve cells using the coaxial multilayer fiber base material to the damaged site of a patient having nerve damage, it is possible to shorten the time until healing for nerve damage, and regenerative medicine It can be used as a cell for use. Examples of such muscle damage and nerve damage include diseases involving nerve damage due to traumatic damage of muscles and nerves such as traffic accidents, and hernia accompanying aging.

  All documents mentioned herein are hereby incorporated by reference in their entirety. The examples described herein are illustrative of embodiments of the invention and should not be construed as limiting the scope of the invention.

1. Experimental materials and equipment Polycaprolactone is manufactured by SigmaAldrich, 1,1,3,3,3-hexafluoro-2-isopropanol (HFIP) is manufactured by Wako Pure Chemicals, and PEDOT / PSS dispersion is Denatron (manufactured by Nagase ChemteX) Polyaniline and polypyrrole manufactured by Sigma Aldrich were used. Reagents used in other experiments were appropriately described in the description of the experimental method.
The core / sheath fiber production system of the electrospinning device Nanon (MECC) was used for spinning. The electron microscope used was a 3D real surface view microscope (VE-9800, manufactured by Keyence).

2. Fiber production method As sheath solution, polycaprolactone (PCL, average molecular weight 85,000) is dissolved in organic solvent 1,1,3,3,3-hexafluoro-2-isopropanol (HFIP) to make 5 wt% solution. Gelatin was mixed so as to be 5% (w / v) with respect to the HFIP capacity. This was used as a sheath solution. A PEDOT / PSS dispersion (Denatron, manufactured by Nagase ChemteX Corporation) stock solution was used as a core solution for forming a conductive polymer. Spinning was performed by an electrospinning method using a core / sheath fiber production system of an electrospinning apparatus Nanon (manufactured by MECC). At that time, the flow ratio of the core solution to the sheath solution was 1:10, the voltage was 25 kV, and the spinning distance was 15 cm (FIGS. 1 and 2).

  The spun fiber was wound up and collected by a drum collector rotating at high speed (2500 revolutions per minute).

  As a comparative sample, a solution (blend solution) in which the sheath solution and the core solution were completely mixed was prepared and spun in the same manner as described above. Using polyaniline and polypyrrole as other conductive polymers other than PEDOT / PSS, except for the same materials as above, adjust each to a 1% (w / v) solution in HFIP as a core solution Used to produce a fiber (FIG. 1). Table 1 shows the average diameter of each fiber prepared by the above method. The core sheath represents a fiber scaffold having a core / sheath dual structure, the blend solution represents a fiber scaffold formed by mixing the core and sheath components, and the sheath solution represents a fiber scaffold formed of the sheath component.

3. Method for evaluating myotube formation efficiency in fiber scaffolds using PEDOT / PSS In order to prevent gelatin contained in the fiber scaffolds from dissolving in the medium, it was essential to stabilize the structure by chemical crosslinking. Therefore, before use, it was exposed to 25% glutaraldehyde vapor for crosslinking. Since there is a possibility of damaging the cells with unreacted glutaraldehyde, the scaffold was immersed in the medium the day before cell seeding and reacted with amino acids and proteins in the medium.

C2C12 cells, which are cell lines derived from mouse myoblasts, were seeded on the fiber scaffold at a density of 5.0 × 10 ⁴ cells / cm ² with respect to the scaffold area. Thereafter, the cells were cultured in a normal growth medium (DMEM, 10% fetal bovine serum, 1% penicillin / streptomycin) in a 37 ° C., 5% CO ₂ incubator for 24 hours. Thereafter, the medium was changed to a differentiation induction medium (DMEM, 2% horse serum, 1% penicillin / streptomycin), and the culture was continued for 5 days, and the differentiation induction medium was changed every other day.

  After completion of the culture, the cells were fixed with 4% paraformaldehyde, the myosin heavy chain was fluorescent immunostained (green), and the differentiation into myotubes was quantitatively evaluated. Cell nuclei were stained with Hoechst (blue) fluorescent reagent (Invitrogen). Statistical analysis was performed using one-way analysis of variance (ANOVA) and Tukey-Kramer method for post-hoc analysis.

Experimental results When the core and sheath fiber scaffolds were used, myotube formation rate was significantly improved compared to the other two conditions. The comparison between the sheath-only fiber and the blended fiber shows that the presence of PEDOT / PSS favors myotube formation, but the core-sheath structure maximizes myotube formation efficiency ( 3 and 4).

Small live cells Using C2C12 cells, the mouse myoblasts described above, by culturing with PEDOT / PSS as a conductive polymer and a coaxial multi-layer fiber substrate made from polycaprolactone and gelatin as a cell adhesion polymer. It was confirmed that the differentiation efficiency was remarkable.

4). Evaluation of electrical properties by surface resistivity measurement The surface resistance value and electrical conductivity of the fiber mat were measured. The results are shown in Table 2. The resistance measuring device Hiresta-UX used in this experiment can measure up to a very high resistance value of ~ 10 ¹⁴ (Ω) by applying a high voltage of 1000 V, and can detect a very weak current. In this experiment, a voltage of 1000 V was applied to all samples and measured. The surface resistance value was highest when “sheath PCL / Gelatin solution only” and “core-sheath material mixture”, and the surface resistance value decreased by 2 to 3 orders of magnitude by introducing the core PEDOT / PSS aqueous dispersion. In addition, the surface resistance value decreased with increasing PEDOT / PSS flow rate. This is thought to be because the PEDOT / PSS layer in the fiber became thicker as the flow rate of the core PEDOT / PSS aqueous dispersion was increased. Although the resistance value of the scaffold manufactured under any condition exceeded 10 ¹⁰ (Ω), which should be called an insulator, the conductivity was improved even though the conductive core layer was covered with an insulator sheath. From this point, the possibility that current reached the core layer at the time of measurement was considered. These values are 8 to 9 orders of magnitude higher in resistance compared with fibers mixed with conductive polymers such as Polyaniline and Polypyrrole. In a system that is simply mixed into the scaffold, the measured value is considered to be high because the conductive polymer is uniformly present on the scaffold surface.

  Furthermore, when the sheath PCL / Gelatin solution concentration was 4 wt (%), it was confirmed that the single-digit surface resistance value was lower than when it was 5 wt (%) (Table 3). As seen from TEM observation, it was considered that the core layer could easily be energized by the thin sheath layer.

Since the voltage applied during the measurement in this experiment is 1000 V, which is very high compared to the potential in the living body (several tens of mV), it is difficult to link the scaffold conductivity with the cellular behavior at present.

5. Comparison of myotube formation on fiber scaffolds made with different core flow rates When producing fibers with a core / sheath dual structure by electrospinning, produced by changing the flow rate of the PEDOT / PSS aqueous dispersion of the core The effects on myotube formation were compared.

  In the case of scaffolds made by changing the flow rate of PEDOT / PSS aqueous dispersion of the core, the cell density after the start of differentiation induction by changing the culture medium to the differentiation-inducing medium is not statistically significant. Showed a tendency to decrease. On the fifth day of differentiation induction, the scaffold with the highest core flow rate of “core flow 0.63 ml / h” showed a tendency that the fusion cell rate, number, and myotube length were all the highest among all scaffold conditions (Fig. 5B, FIGS. 6A-C).

  Myotube formation can be confirmed from day 0 and day 1 of differentiation induction, and myotube formation is the highest at `` core flow rate 0.5 ml / h '' and `` core flow rate 0.63 ml / h '' on induction days 0 to 3 Became high. On the fifth day of differentiation induction, the myotube shape rate was the same at “core flow rate 0.5 ml / h” and “core flow rate 0.63 ml / h”. In addition, there was no significant effect on myotube formation between the core flow rates of 0 and 0.33 ml / h, and myotube formation efficiency increased at 0.50 ml / h or more, so a certain amount of core PEDOT / PSS water dispersion It was shown that myotube formation was promoted when fluid was introduced (FIG. 6B).

6). Evaluation of myotube formation in scaffolds prepared by changing the sheath PCL (polycaprolactone) / gelatin solution concentration Next, myotube formation was evaluated when the thickness of the sheath layer was changed.

  In the spinning of core / sheath dual structure fibers, the thickness of each layer has been shown to be related to the concentration of the polymer solution (Elahi, MF et al., J. Bioeng. Biomed. Sci. 3, 1-14 2013 ). Assuming that C2C12 cells on the surface of the sheath layer and the internal PEDOT / PSS are influencing each other, the thinned sheath layer makes the surface cells more susceptible, that is, promoting myotube formation We assumed that it would be easier.

  The results of myotube formation evaluation are shown in FIGS. There was no significant difference in the cell density of each scaffold throughout the differentiation induction period. On the other hand, as the sheath PCL / gelatin solution concentration decreased, the area ratio, myotube number, and fused cell rate positive for myosin heavy chain increased, and the maximum when the sheath PCL / gelatin solution concentration was the lowest 3 wt (%). The tendency to become was seen. Regarding myotube length, there is no significant difference between the sheath concentration of 4 wt (%) and 3 wt (%) on the 5th day of differentiation induction. However, when efficiently creating skeletal muscle that functions as an entire tissue, it is considered important to have a larger area ratio and number of myotubes as evaluation items for myotube formation, so a 3 wt (%) scaffold is myotube formation. We thought that it was excellent as an induction scaffold. In addition, when the sheath concentration was 4 wt (%), the surface resistance of the scaffold was reduced by an order of magnitude compared to the sheath concentration of 5 wt (%). This is thought to have facilitated myotube formation.

Summary From the above results, when myoblasts are cultured as a scaffold with a core / sheathed double layer encapsulating a conductive polymer as the core and a cell-adhesive gel on the outside, induction of myotube formation It was confirmed in this study that it has an effect.

  Since this sheath structure has a high surface resistance value, it is considered that the sheath solution does not act on the cultured cells by directly contacting the conductive polymer. Further, since this action is promoted by increasing the core structure or reducing the sheath structure, it is considered that the effect of the conductive polymer of the core structure affects the performance of the myotube formation action. .

Claims (11)

  A culture substrate for inducing and / or proliferating cells, comprising a conductive polymer layer containing a conductive polymer and a cell adhesion polymer layer containing a cell adhesion polymer. A base material characterized by being a multilayer film. The cell is a cell having a fiber-forming ability;
The multilayer film encloses the fiber-like conductive polymer layer, and at least one cell adhesion polymer layer is laminated coaxially with the conductive polymer layer in the outermost layer, whereby a core / sheath structure or The substrate according to claim 1, wherein the substrate is a multilayer fiber having a coaxial multilayer structure. The conductive polymer is selected from PEDOT / PSS [poly (3,4-ethylene dioxythiophene) / poly (4-styrenesulfonic acid)], polyaniline or polypyrrole;
The cell adhesion polymer is
A cell adhesion polymer comprising polycaprolactone, polylactic acid, polyglycolic acid, or a copolymer thereof;
A polymer for general culture equipment comprising polymethyl methacrylate or polystyrene; and
A bio-derived polymer comprising collagen or gelatin;
The base material according to claim 1, wherein the base material is at least one selected from the group consisting of:   4. The cell according to claim 1, wherein the cell having the ability to form a fiber is selected from a myoblast, a nerve cell, a cell line or a primary cell thereof, or a model cell. 5. Base material.   A method of promoting differentiation induction and / or proliferation of a cell, comprising a multilayer film comprising a conductive polymer layer containing a conductive polymer and a cell adhesion polymer layer containing a cell adhesion polymer as a base material. A method comprising culturing. The cell is a cell having a fiber shape forming ability,
The base material is a coaxial multilayer fiber including a fiber-like conductive polymer layer and having at least one cell adhesion polymer layer as an outermost layer;
The method according to claim 5, wherein the cells are cultured on the substrate. The conductive polymer is selected from PEDOT / PSS [poly (3,4-ethylene dioxythiophene) / poly (4-styrenesulfonic acid)], polyaniline or polypyrrole;
The cell adhesion polymer is
A cell adhesive polymer comprising polycaprolactone, polylactic acid, polyglycolic acid or a copolymer thereof;
A polymer for general culture equipment comprising polymethyl methacrylate or polystyrene; and
A bio-derived polymer comprising collagen or gelatin;
The method according to claim 5, wherein the method is at least one selected from the group consisting of:   The cell having a fiber shape-forming ability is selected from myoblasts, nerve cells, cell lines or primary cells thereof, or model cells thereof, according to any one of claims 5 to 7, The method described.   A cell, characterized by being cultured using the substrate according to any one of claims 1 to 4.   A cell, characterized by being cultured using the method according to any one of claims 5 to 8.   The cell according to claim 9 or 10, wherein the cell is a cell for cell engineering and / or regenerative medicine.