KR102200859B1 - Method for manufacturing cell-laden scaffold for tissue regeneration using cell electrospinning - Google Patents

Abstract

The present invention relates to a method of manufacturing a cell scaffold for tissue regeneration using cell electrospinning, and the like, wherein the present inventors have made intensive research to develop a stacked structure having an arrangement using cell electrospinning, and as a result, alginate carrying muscle cells A cell scaffold having a fiber structure arranged in one direction can be produced by electrospinning a cell scaffold in which cells are uniformly distributed in the arrayed fibrous structure, and the cell scaffold is the direction in which the cell growth is aligned. It was confirmed that it can be induced by, compared to a three-dimensional cell support without alignment, the alginate structure carrying cells induces uniform cell distribution and more effective direction of cell growth through three-dimensional arrayed fiber bundles. Therefore, it was confirmed that the cell elongation, alignment, and differentiation were increased, and the shape of muscle cells, effectively arranged cell structure, and degree of differentiation were confirmed through fluorescence staining. It has been confirmed that it plays a large role in regenerating tissues, and it is expected that the cell scaffolds produced through the present invention can be effectively used to regenerate tissues/organs having alignment.

Description

Method for manufacturing cell-laden scaffold for tissue regeneration using cell electrospinning {Method for manufacturing cell-laden scaffold for tissue regeneration using cell electrospinning}

The present invention relates to a method of manufacturing a cell support for tissue regeneration using cell electrospinning, and more specifically, a cell-containing bioink prepared by mixing cells in a biocompatible polymer mixed solution with parallel electrodes. ) By electrospinning cells (cell eletrospinning; CE) to prepare a scaffold, and to a cell scaffold for tissue regeneration manufactured by the method of manufacturing the same.

Tissue engineering is a field in which several disciplines aim to develop biological products that regenerate, maintain, or improve the function of tissues using the principles of life science and engineering. A typical method is a procedure for artificially forming a tissue by separating and culturing cells from a tissue to be regenerated, and inoculating the cells in an appropriate biomaterial to amplify and culture.

Such a procedure requires a suitable cell support that makes it easy to deliver cells to the required area, can serve as a mechanical aid to the growth of tissues, and can function as a new tissue that can function. Such a scaffold must have an appropriate microstructure to allow cells to proliferate and create a unique matrix, and has many pores interconnected in three dimensions, so that cells can grow inward through these pores, and provide nutrients necessary for cell growth. Must be able to supply. In addition, it should be a biodegradable material that is not toxic and can be completely degraded and disappeared in vivo after its function as a support is terminated.

To improve the performance of artificial tissues, recent artificial tissues can hold growth factors and cells in a desired location, distribute cells evenly, and effectively transport growth factors. techniques) have been widely applied in various tissue regeneration fields, but when cells are embedded in bioink, it is difficult to induce cell growth in the desired 3D geometry. In addition, shearing of cells in the 3D printing process may reduce the number of extruded cells, and may become an obstacle to inducing cell-cell interactions. In this concern, cells without external stimuli or micropatterns have less cell-cell interactions and grow in random shapes and omni-directional arrangements. Therefore, there is a need for an environment that facilitates cell migration and micropattern-inducing cells in an ordered arrangement for a microenvironment.

On the other hand, the human body is composed of a number of tissues with an arranged fiber bundle structure, examples of which include nerves, tendons, and muscles. Cells must be cultured and differentiated in an arrayed form in order to regenerate a tissue having such an ordered structure. Therefore, many research teams have been trying to produce a cell scaffold having a fibrous structure arranged in various ways in order to cultivate cells in an arrayed form. However, although it has been frequently used to fabricate an arrayed fiber structure such as an electrospinning process or a lithography process, it is generally impossible to stack, and there are limitations such as non-uniform distribution of cells during cell seeding, and poor and time-consuming cell penetration. It was a situation with a back.

However, as a technology to solve this, a cell electrospinning (CE) process has been developed to provide guidelines for fine patterns, homogeneous cell distribution, and improved nutrient accessibility. CE is a cell-supported fiber manufacturing method in which a polymer solution containing living cells is drawn up to a fiber diameter of about several hundred nanometers by using electric force, and CE shares the characteristics of electrospraying and dry spinning of existing solutions. do. Since this process does not require coagulation chemistry, organic solvents, or high temperatures to produce fibers in solution, it is possible to induce cell growth through uniform cell distribution and fiber structure in three dimensions through biocompatible processes. The development of a stacked structure with an array of structures has not been reported, so the possibility of application to various fields such as regeneration of an arrayed tissue remains a question, and a lot of research on this is required.

Korean Registered Patent Publication 10-1449645

The present invention comprises the steps of (a) mixing alginate and polyethylene oxide (PEO) to prepare a biocompatible polymer mixed solution; (b) preparing a cell-containing bioink by mixing the cells in the biocompatible polymer mixed solution prepared in step (a); And (c) producing a cell scaffold by performing cell electrospinning (CE) with the cell-containing bioink prepared in step (b) with parallel electrodes. It is an object of the present invention to provide a method for producing a cell scaffold.

In addition, an object of the present invention is to provide a cell support for tissue regeneration, characterized in that the cells are arranged in a living state and have a cell-containing nanofiber bundle structure.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems that are not mentioned can be clearly understood by those of ordinary skill in the technical field to which the present invention belongs from the following description. There will be.

In order to achieve the object of the present invention as described above, the present invention comprises the steps of (a) mixing alginate and polyethylene oxide (PEO) to prepare a biocompatible polymer mixed solution; (b) preparing a cell-containing bioink by mixing the cells in the biocompatible polymer mixed solution prepared in step (a); And (c) producing a cell scaffold by performing cell electrospinning (CE) with the cell-containing bioink prepared in step (b) with parallel electrodes. It provides a method for producing a cell scaffold.

In one embodiment of the present invention, in the step (a), the biocompatible polymer mixed solution may have a weight ratio of alginate/PEO of 0.5 to 2.

In another embodiment of the present invention, the biocompatible polymer mixed solution has a surface tension of 38 to 48 mN/m, an electrical conductivity of 2.9 to 3.9 μS/cm, or, depending on the weight ratio of the alginate/PEO. Complex viscosity may be 1~15Pa ㆍ S.

In another embodiment of the present invention, in the step (c), the cell electrospinning has a nozzle-electrode spacing of 12 to 14 mm, an electric field range of 0.050 to 0.100 kV/mm, and an electrode-electrode spacing of 5 to 30 mm, Alternatively, a cell scaffold can be prepared by setting the electrospinning time to 60 to 300 seconds.

In addition, the present invention is a tissue regeneration cell support prepared by the above manufacturing method, wherein the tissue regeneration cell support has a cell-containing nanofiber bundle structure in which cells are arranged in a living state, for tissue regeneration Provides cell support.

In one embodiment of the present invention, the arrayed cell-containing nanofiber bundle structure may have a full width at half maximum (FWHM) of 10 to 40°.

In another embodiment of the present invention, the cell may have an aspect ratio of F-actin of 4 to 6, or an orientation of F-actin of FWHM of 0 to 20°.

In another embodiment of the present invention, the cell scaffold for tissue regeneration may express a myosin heavy chain (MHC).

In another embodiment of the present invention, the MHC has an MHC positive index of 90 to 100%, a fusion index of 30 to 90%, or a maturation index of 30 to 70%. I can.

In another embodiment of the present invention, the cell scaffold for tissue regeneration may have a sarcomeric structure.

In another embodiment of the present invention, the cell scaffold for tissue regeneration has a cell viability of 80 to 95%, a FWHM of 10 to 40°, a fiber density of 120 to 240/μm ² , or a tensile strength of 30 to 35 MPa, or relative cell proliferation may be 100% to 122%.

In another embodiment of the present invention, the cell support for tissue regeneration may be for skeletal muscle tissue regeneration.

In another embodiment of the present invention, the cells may be myoblasts.

As a result of intensive research to develop a stacked structure with alignment using cell electrospinning, the present inventors produced three-dimensional fiber structures and bundles arranged by applying an electrospinning process after supporting cells in alginate, a natural polymer. A possible process was developed, and conditions for spinning the alginate bio-ink carrying cells were established, and the alginate carrying the muscle cells was electrospun to produce a cell scaffold in which cells were uniformly distributed in the arranged fibrous structure.

Through this, the present inventors can produce a cell scaffold having a fibrous structure arranged in one direction, and it has been confirmed that the cell scaffold can induce cell growth in an aligned direction, which is a three-dimensional cell scaffold without alignment. In contrast, it was confirmed that the alginate structure carrying cells increased cell elongation, alignment, and differentiation because it can induce uniform cell distribution and more effective cell growth direction through three-dimensional arrayed fiber bundles. Through staining, the shape of muscle cells, effectively arranged cell structure, and degree of differentiation were confirmed, and it was confirmed that the cell scaffold produced through the present invention plays a large role in regenerating the arranged tissue.

Therefore, it is expected that the cell scaffolds produced through the present invention can be effectively used to regenerate tissues/organs having alignment.

1A is a diagram showing a schematic diagram of a cell electrospinning (CE) cell structure process of an arranged alginate fiber structure according to Example 1-5.
1B is a diagram showing an optical image and electric field strength expressed by simulation.
1C is an optical image of electrospinning using pure alginate and alginate/PEO solution, an SEM image according to Examples 1-4, and a diagram showing a molecular chain.
Figure 2a is a diagram showing surface tension, electrical conductivity, composite viscosity (n = 3), and fiber diameter (n = 50) for alginate (2% by weight) having different weight fractions of PEO (0-4% by weight). to be.
Figure 2b is a diagram showing the SEM image in gray scale according to Example 1-4 showing the fiber structure (fiber alignment and fiber distribution) according to the composition of the biocompatible polymer mixed solution (n = 50).
3A is a diagram showing a process window of electrospinnability showing beads (X), beads and fibers (Δ) and fibers (○) (n = 5).
3B is a view showing an SEM image of an electrospun fiber according to Example 1-4 prepared using a 10-14mm nozzle-electrode distance and a 0.050-0.075kV mm ^-1 electric field.
3C is a diagram showing SEM and survival/kill images according to Examples 1-4 of cell-containing fibers prepared using 0.050-0.075 kV mm ^-1 at a 14 mm nozzle-electrode distance.
3D is a graph showing the cell viability of FIG. 3C (n=5).
3E is a diagram showing SEM images of electrospun fibers before and after crosslinking.
4A is a diagram showing SEM images and orientation distributions according to Examples 1-4 for various electrode-to-electrode distances.
4B is a graph showing the full width at half maximum (FWHM) and fiber density with respect to the electrode-to-electrode distance obtained in FIG. 4A (n = 50).
4C is a diagram showing an SEM image and orientation distribution according to Examples 1-4 with respect to electrospinning time.
Figure 4d is a graph showing the FWHM versus the electrospinning time (n = 50) obtained in Figure 4c.
Figure 5a is a diagram showing a cell printing (Cell printing; CP) cell support and electrospinning (cell electrospinning (CE)) cell support in schematic, optical, survival / death images.
Figure 5b is a graph showing the cell viability before and after CaCl ₂ crosslinking in the CP cell support and the CE cell support (n = 3).
5C is a diagram showing an optical image and a stress-strain curve (n = 5) of a tensile test according to Examples 1-7 of CP cell scaffolds and CE cell scaffolds.
Figure 5d shows the survival / death images of the CP cell scaffold and the CE cell scaffold on days 1 and 7.
Figure 5e is a graph showing the cell viability of the CP cell support and the CE cell support (n = 3).
Figure 5f is a graph showing the relative cell proliferation of the CP cell support and the CE cell support (n = 6).
5G shows CE cell scaffolds represented by optical images, survival/kill images, DAPI/phalloidin images, and MHC-stained images.
6A shows a schematic of a cell printing (CP) and cell electrospinning (CE) process supplemented with a polycaprolactone (PCL) strut (hereinafter, referred to as a PCL strut).
Figure 6b shows an optical image of the CP cell support and the CE cell support according to the presence or absence of the PCL support.
6C is a graph showing the stress-strain curves of printed alginate, electrospun alginate, and PCL braces (n = 5).
6D is a diagram showing a cross section of a cell scaffold prepared with a PCL brace as DAPI/phalloidin.
6E is a diagram showing a muscle-mimicking electrospun PCL bundle structure in a schematic, optical, and SEM image according to Examples 1-4 on the surface and in cross section.
Figure 7a is a diagram showing the survival / death images of the CP cell support and the CE cell support on days 1 and 7 (n = 3).
7B is a graph showing the cell viability of the CP cell support and the CE cell support on days 1 and 7 (n = 3).
7C is a diagram showing the cell morphology obtained by the SEM image according to Example 1-4 on the 7th day.
7D is a diagram showing the cell morphology obtained by DAPI / phalloidin staining on the 7th day.
Figure 7e is a diagram showing a diagram of the F-actin aspect ratio and F-actin orientation and analysis accordingly (n = 30).
7F is a diagram showing fluorescence images of DAPI / MHC staining on days 7, 14 and 21.
Figure 7g is a diagram showing the MHC positive index and MHC analysis (n = 30).
Figure 7h is a diagram showing a schematic and MHC analysis of the fusion index (n = 30).
Figure 7i is a diagram showing a schematic and MHC analysis of the maturity index (n = 30).
7j is a diagram showing optical and DAPI/sarcomeric α-actin staining images 21 days after culture according to Examples 1-8.

As a result of the inventors' intensive research to develop a stacked structure with arrangement using cell electrospinning, the inventors produced a cell-containing bioink containing myoblasts in alginate, As a result of using high voltage direct current, spinning on parallel electrodes arranged in parallel to produce a three-dimensional cell-carrying fiber structure, and making the myoblasts included under the influence of the alginate fiber structure also have an arrayed shape. It was confirmed that a cell scaffold having a fibrous structure arranged in one direction can be produced, and it was confirmed that the cell scaffold can induce cell growth in an aligned direction. This is compared to a three-dimensional cell scaffold without alignment, It was confirmed that the alginate structure increased cell elongation, alignment, and differentiation because it can induce uniform cell distribution and more effective direction of cell growth through three-dimensional arrayed fiber bundles. The shape, effectively arranged cell structure, and degree of differentiation were confirmed, and it was confirmed that the cell scaffold produced through the present invention plays a large role in regenerating the arranged tissue, and the present invention was completed.

Hereinafter, the present invention will be described in detail.

In an embodiment of the present invention, it is confirmed that the directionality of the fibers is directly affected by the electric field in the spinning process, and the fibers are arranged and spun between two electrodes arranged in parallel, and the pure alginate solution is polyanion. When it was confirmed that spinning was impossible due to the repulsive force of, it was confirmed that molecular entanglement required for electrospinning was formed by adding polyethylene oxide (PEO) to enhance electrospinning properties. See example 2).

In another embodiment of the present invention, in order to know the composition ratio of a solution capable of spinning, various ratios of solutions were tested through comparison of electrospinability and fiber alignment in a cell-containing bioink mixed with alginate having various PEO fractions. As a result, it was confirmed that the A2P3 solution and bio-ink in which 3% by weight of PEO was added to 2% by weight of alginate were suitable (see Example 3).

In another embodiment of the present invention, manufacturing conditions were established to produce a structure having the highest alignment using A2P3 solution and bio-ink. As conditions affecting electrospinning, experiments were conducted with various ranges of electric field, nozzle-to-electrode distance, electrode-to-electrode distance, and radiation time. As a result, when the distance between the nozzle and the electrode was 14 mm and the electric field was applied at 0.075 kV/mm, high alignment and high cell viability were confirmed.After that, the distance between the electrodes was 30 mm and the spinning time was 180 s. It was confirmed that the structure could be stably manufactured (see Example 4).

In another embodiment of the present invention, a CE cell scaffold was prepared, and as a result of confirming the in vitro cell activity according to Examples 1-9 on the CE cell scaffold, a high cell viability was confirmed (see Example 5). ).

In another embodiment of the present invention, in order to increase the mechanical stability of the CE cell support, as a result of supplementing the PCL brace, it was confirmed that the cell-containing fiber bundles in the CE cell support including the PCL brace were mechanically more stable (Example 6 Reference).

In another embodiment of the present invention, as a result of confirming the cell activity in the CE cell scaffold including the PCL brace, it was confirmed that cell elongation and sorting are more advantageous by the alignment induction on the scaffold surface, myosin heavy chain; MHC) and sarcomeric α-actin staining, it was confirmed that MHC and sarcomere structures were expressed (see Example 7).

Accordingly, the present invention comprises the steps of (a) mixing alginate and polyethylene oxide (PEO) to prepare a biocompatible polymer mixed solution; (b) preparing a cell-containing bioink by mixing the cells in the biocompatible polymer mixed solution prepared in step (a); And (c) producing a cell scaffold by performing cell electrospinning (CE) with the cell-containing bioink prepared in step (b) with parallel electrodes. It provides a method for producing a cell scaffold.

The term 'alginate (alginate), to be used in the present invention, as obtained from the brown algae with anionic properties, good processability with rapid gelation by the addition of cations such structural similarity with the extracellular matrix, and non-toxic and Ca ^{2 +} For this reason, it has been widely used in tissue engineering. Because of these properties, alginate can also be applied as a cell-containing bioink, but is not limited thereto.

In the present invention, in order to form stable cell-containing microfibers without beads, the weight% of alginate in the bioink for cell electrospinning may be selected from 1% to 3% by weight by a method known in the art. Weight%, preferably 2% by weight alginate may be optimally selected, but is not limited thereto.

The term "polyethylene oxide (polyethelene oxide; PEO)" used in the present invention _{is, - [- CH 2 CH 2} O-] - n means a linear polyether represented by the formula. Depending on the polymerization conditions, a molecular weight of 200 to 10000 or more can be obtained, and a variety of different properties can be obtained from liquid to hard waxy solid. All are well soluble in water due to the hydrophilicity of the ether group. It is soluble in most organic solvents including aromatic hydrocarbons and insoluble in aliphatic hydrocarbons. In general, it has good chemical resistance and does not cause decomposition to around 300°C. Flexibility is much better than polyoxymethylene and polypropylene oxide. High molecular weight ones have good crystallinity, high tensile strength, and do not have hygroscopicity. Block and graft copolymers of polyethylene oxide are also being synthesized. In addition to being used in a surface active agent and a neutral detergent, it may be used in pharmaceuticals, cosmetics, and also as a plasticizer, but is not limited thereto.In the present invention, PEO is a carrier polymer, reducing surface tension and electrical conductivity, and more preferably Molecular entanglement required for electrospinning may be formed, but is not limited thereto.

In the present invention, in order to increase the low electrospinability of alginate, in addition to polyethylene oxide, various processing agents known in the art may be used, for example, processing of activated carbon, flame retardant processing agent, softening agent, or oil processing agent. The best can be, but is not limited thereto.

According to a specific embodiment of the present invention, in the step (a), the weight ratio of alginate/PEO in the biocompatible polymer mixed solution may be adjusted by a method known in the art, and 0.25 to 3, preferably 0.5 to 2 However, it is not limited thereto. In addition, the biocompatible polymer mixed solution may have a surface tension of 30 to 50 mN/m, preferably 38 to 48 mN/m, and an electrical conductivity of 2.5 to, depending on the weight ratio of alginate/PEO. 4.5μS / cm preferably 2.9 to may be a 3.9μS / cm, or complex viscosity (complex viscosity) is may be a 0.5 - 25Pa and S preferably 1 ~ 15Pa and S, but not limited thereto.

The term'surface tension' as used in the present invention means a tension acting on a free surface of a liquid to reduce the surface. Since molecules near the liquid surface have higher potential energy than molecules in the liquid, the liquid has a surface energy proportional to the surface area, and this creates a surface tension.

The term'electrical conductivity' used in the present invention means that a current flows when a constant electric field is applied to a conductor (or a voltage is applied between both ends), and the current density is proportional to the electric field. Electrical conductivity means its proportionality constant.

The term'complex viscosity' used in the present invention is also called complex viscosity, and is a mathematical expression in which the viscosity is the sum of the real and imaginary parts. The real part is usually called kinetic viscosity, and the imaginary part is related to the real part of the complex inspection modulus.

In the present invention, the parallel electrodes are parallel electrodes for manufacturing by aligning nanofibers by cellular electrospinning, and anisotropic array fibers are deposited on the parallel electrodes under an electric field applied between the nozzle and the parallel electrodes. That is, the electrospun fibers may be aligned between the parallel electrodes through the torque induced in the electric field generated by the induced dipole moment due to the electric field direction and shape coefficient of the parallel electrodes, but is not limited thereto.

The term'cell eletrospinning (CE)' used in the present invention refers to a method for producing cell-supported fibers in which a polymer solution containing living cells is drawn up to a fiber diameter of about several hundred nanometers using electric force, CE shares the properties of fiber electrospraying and conventional solutions dry spinning. Since this process does not need to use coagulation chemistry or high temperature to generate yarn from solution, the process is particularly suitable for producing fibers using large and complex molecules, thus ensuring uniform cell distribution in three dimensions and cell growth through fiber structure. You can induce. In other words, it refers to a technology that enables the guidance of cell growth through micro/nano fiber structures using various natural polymers and living cells. As a multifunctional tool to create micro/nano fiber structures with controllability of topological signals, The fabricated micro/nano fibers are similar to the structural geometry of the extracellular matrix (ECM), and are therefore widely used to regenerate various tissues. Successful electrospinning of human brain (1321N1) cells in PDMS solution is possible, and under a 0.09 kV mm ^-1 electric field, cells can exhibit a survival rate of 67.6% and cell proliferation for 9 days. In addition, this technique can regenerate different cells such as neuroblastoma, adipose stem cells, and cardiomyocytes using a variety of solutions, and electrospun cardiac patches can also be made from major neonatal cardiac myocytes. , Without being limited thereto, the processed heart patch may represent a structured myofibril represented by MyBP-C, sarcomeric α-actin, connexin-43 and myomesin, but is not limited thereto. Through electrospinning using osteoblast-like cells (MG63) and alginate/polyethylene oxide (PEO)/lecithin solution, the extracellular matrix (ECM) structure can be mimicked, and a 0.16 kV mm ^-1 electric field is used. Thus, cells are electrospun with more than 80% cell viability, and cell proliferation and osteogenic differentiation can be exhibited. CE technology may belong to the final examination of human phase 1 clinical trials, but is not limited thereto.

According to a specific embodiment of the present invention, the cell electrospinning has a nozzle-electrode spacing of 10 to 15 mm, preferably 12 to 14 mm, an electric field range of 0.025 to 0.125 kV/mm, preferably 0.050 to 0.100 kV/mm, and an electrode- A cell scaffold may be manufactured with a spacing between electrodes of 3 to 40 mm, preferably 5 to 30 mm, or an electrospinning time of 50 to 420 seconds, preferably 60 to 300 seconds, but is not limited thereto.

In addition, the present invention is a tissue regeneration cell support prepared by the above manufacturing method, wherein the tissue regeneration cell support has a cell-containing nanofiber bundle structure in which cells are arranged in a living state, for tissue regeneration Provides cell support.

According to a specific embodiment of the present invention, the arrangement of the cell-containing nanofiber bundle structure may have an FWHM of 8 to 100° and preferably 10 to 40°, and the cells have an aspect ratio of F-actin. A 2 to 8, preferably 4 to 6, or the orientation (orientation) of F-actin is 0 to 30 ° FWHM may be preferably 0 to 20 °, but is not limited thereto.

The term'aspect ratio' used in the present invention refers to the ratio of the length of the short axis to the short axis in a two-dimensional model, and in the present invention, cell elongation can be confirmed through the aspect ratio. However, it is not limited thereto.

The term'orientation' used in the present invention means an angular relationship between the axis of a crystal and the basic axis of the system. For a plane, it can be expressed as the angular relationship between the plane's vertical line and the basic axis of the system. In the present invention, the orientation may confirm cell alignment through FWHM, but is not limited thereto.

According to a specific embodiment of the present invention, the cell scaffold for tissue regeneration may express a myosin heavy chain (MHC) or a sarcomeric structure, but is not limited thereto.

The term'myosin heavy chain (MHC)' used in the present invention is a kind of protein constituent of myosin with a molecular weight of about 220,000. The C-terminal side is the rod region of the alpha helix responsible for the filament-forming ability of myosin and is called the tail. The N-terminal side is a globular region, called the head of myosin, and has ATPase activity, myosin L chain and actin binding site. The two H chains form two-headed myosin molecules by winding coils with each other at the tail. In general, the function of myosin is controlled by the myosin L chain, but depending on the type of myosin, the activity is controlled by phosphorylation of the tail. In the present invention, MHC is a protein required for the formation of myotubes, and it can be confirmed that it is expressed to indicate myogenesis, but is not limited thereto.

In the present invention, the MHC has an MHC positive index of 80 to 110%, preferably 90 to 100%, a fusion index of 20 to 100%, preferably 30 to 90%, or maturation. The maturation index may be 20 to 80%, preferably 30 to 70%, but is not limited thereto.

The term'sarcomeric structure' used in the present invention refers to the structure of a unit in which myofibrils in the rhabdomyos are repeated. It represents between the neighboring Z classes and can be made in the order of Z class-I vs.A vs.I vs.Z class. The three tissues that transmit stimuli from nerves to the myofibrils are also located in the vicinity of the Z half, and the T-tube of the transmission system and the muscle vesicles are also repeated like eradication. The length of the apex is shortened upon contraction, but in vertebrates, it is usually 2-3 μm at relaxation. In the present invention, in order to confirm the mature differentiation stage of the myotube, the root structure can be confirmed, but is not limited thereto.

According to a specific embodiment of the present invention, the cell scaffold has a cell viability of 80-100%, preferably 80-95%, a fiber distribution FWHM of 8-100°, preferably 10-40°, and a fiber density of 90-250 /μm ² Preferably 120 to 240 / μm ² , tensile strength is 20 to 40 MPa, preferably 30 to 35 MPa, or relative cell proliferation is 95% to 130%, preferably 100 to 122% However, it is not limited thereto.

The term'full width at half maximum (FWHM)' used in the present invention is a term representing the width of a function and is defined as the difference between two independent variable values that are half of the maximum value of the function. The full width at half maximum is used to indicate the duration of a pulse in signal processing and the bandwidth of a signal used in communication. In the present invention, through the FWHM, it is possible to determine the ordered structure of the fiber made of CE, and the lower the value, the better the alignment, but is not limited thereto.

The term “tensile strength” used in the present invention is also referred to as tensile strength, and is one of values indicating the mechanical strength of a material. Pull the rod-shaped test piece and obtain the tensile strength from the applied load and the shape of the deformation of the test piece. When a load is applied to the test piece, the test piece stretches in proportion to the load, and eventually, it deviates from the proportional relationship. At a certain elongation value, the maximum load M divided by the original cross-sectional area of the test piece is the tensile strength, indicating the maximum load that can be supported in a unit area. It can be used for the adhesion test of decorative electroplating on plastic, but is not limited thereto. In the present invention, it is possible to determine whether or not the fabricated cell scaffold has mechanical stability through the tensile strength, but is not limited thereto.

In addition, in the present invention, the cell support for tissue regeneration may be for skeletal muscle tissue regeneration, and the cells may be myoblasts, but are not limited thereto.

The term'skeletal muscle' used in the present invention is a muscle that is attached to the movable part of the skeleton and controls the movement, and is a transverse pattern muscle and a voluntary muscle. One skeletal muscle is composed of a number of muscle fibers and connective tissue, and the movement of the muscle performs contractile movement according to the direction of the muscle fiber. In the skeletal muscle, motor, sensory, and autonomic nerves are distributed. The skeletal muscles that can regenerate tissue include, for example, a muscle with only one root head, a muscle that exerts strong power, such as a monoceps, two or more biceps, a biceps brachii, biceps femoris, triceps brachial, triceps lower arm, quadriceps thigh. There may be multiple abs, jaw abs, scapular hyoid muscle, abdominal rectus muscle, etc., in which the middle of the muscle is interrupted by tendons, but are not limited thereto.

The term "myoblast" used in the present invention is a muscle cell in an undifferentiated state and constitutes a muscle node. At first, it forms a cylindrical shape, but by mitosis, several clusters are formed in parallel. In skeletal muscle fibers, myofibrils are formed in the myoblasts (monocytes), followed by horizontal patterns. The contractile function also begins on or before this myofibrillar formation. The number of myofibrils in the ventricular muscle fibers is small, and the myofibrils increase as they occur later. The number of muscle fibers itself is determined during exhaustion, after which it becomes enlarged but does not proliferate, but there are exceptions such as levator ani.

Hereinafter, preferred embodiments are presented to aid in understanding the present invention. However, the following examples are provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[ Example ]

Example 1. Materials and methods

1-1. material

Sodium alginate (LF10 / 50, FMC BioPolymer, Drammen, Norway) was provided by Pharmaline (Suwon, South Korea). PEO (Mw = 900000), calcium chloride (CaCl ₂ ) and PCL (Mw = 45 000) were purchased from Sigma-Aldrich (St. Louis, MO). C2C12 myoblasts (CRL-1772) used for CE and CP were purchased from ATCC (Manassas, VA).

1-2. Alginate / PEO Preparation of solution and bioink

To characterize the alginate/PEO solution 2% by weight alginate and 0-4% PEO were dissolved in tri-distilled water three times. The solution was stirred at room temperature for several days using a magnetic bar. As a bioink, a 2% by weight alginate/3% by weight PEO solution was selected, and C2C12 was added in 5 × 10 ⁶ cells mL ^-1 .

1-3. Alginate / PEO Solution characterization

The following characterization of the alginate/PEO solution was carried out at room temperature. For surface tension measurement, the glass capillaries were immersed in the alginate/PEO solution. After 1 hour, the glass capillary was captured using a digital camera (Canon EOS 450D; Canon, Japan). Then, the surface tension was measured by contact angle measurement. To measure the conductivity of the alginate/PEO solution, a pH/conductivity meter (Consort Bvba, Belgium, Turnhout, Consort C861) was used.

Rheological measurements were performed to obtain a complex viscosity (n *). Using a rotary rheometer (Bohlin Gemini HR Nano; Malvern Instruments, Surrey, UK) cone and plate structure (4° cone angle, 40 mm diameter, and 150 μm gap) with different PEO weight fractions (1-4% by weight). Alginate (2% by weight) was tested. The dynamic frequency sweep was performed from 0.1 to 10 Hz using 2% strain and 24°C in the linear viscoelastic region.

1-4. Analysis of Surface Morphology

For microscale imaging, the electrospun fiber was attached to a double-sided carbon tape and placed on a pedestal for Au coating. Thereafter, the fiber morphology of the sample was captured using a scanning electron microscope (SEM; SNE-3000M; SEC Inc., Suwon, South Korea). Fibers were analyzed in terms of fiber diameter, alignment and density using ImageJ software.

1-5. Cell electrospinning eletrospinning ; CE) process and cell printing process

For the CE process, the prepared bio-ink was mounted on a syringe pump (KDS 230, NanoNC, Inc.) at a flow rate of 0.25 mL/h. Subsequently, 10.5kV high voltage direct current was applied using a high voltage power source (SHV300RD-50K, Convertech). The bioink was electrospun onto two cylindrical electrodes (diameter: 360 μm) for 3 minutes. Video of this process was recorded using a digital camera (Nikon D3100, Nikon, Japan) and provided as supporting information. As an application, PCL braces were mounted between the electrodes.

In the cell printing process, bioink was distributed using a 3D robot (DTR3-2210-T-SG, DASA Robot, South Korea). An air pressure of 120 kPa and a nozzle movement speed of 10 mm s ^-1 were applied. To apply to the PCL brace, PCL was melted at 100° C. in a heating cartridge, and then PCL was drawn with the brace using 340 kPa. After a while, the bio-ink is dispensed on PCL braces with a 100 μm gap using the same manufacturing conditions as described above. All samples prepared using CE and cell printing were crosslinked with 2 wt% CaCl ₂ in Dulbecco's modified Eagle's medium (DMEM) with high glucose for 2 minutes. The sample was then rinsed twice using tris-buffered saline.

1-6. Cell count

In order to calculate the number of cells, C2C12 cells were included in a bio-ink of 5 × 10 ⁶ cells mL ^-1 and applied to the CP and CE processes according to Example 1-5. For CE, the conditions obtained to prepare the CE scaffold were used, but the electrospun fibers were collected on a 2D electrode for cell counting. In the case of cell printing, a 3D bioprinter was used to draw a brace under the same manufacturing conditions as the CP scaffold. After treatment with trypan blue solution (Sigma-Aldrich), the sample was observed using an optical microscope (CKX41, Olympus) to count the number of cells.

1-7. Tensile Property Measurement

The stress-strain curve was obtained using a micro-tensile tester (Toptech 2000; Chemilab, Suwon, Korea). Before the test, the sample was freeze-dried and uniaxially stretched at 0.05 mm s ^-1 .

1-8. In vitro cell culture

C2C12 myoblasts were cultured using 10% fetal bovine serum (Gemini Bio-Products, Sacramento, CA, USA) containing DMEM with high glucose and 1% antibiotics (Antimycotic, Cellgro, Manassas, VA). . Samples were stored in an incubator supplemented with 5% CO ₂ under conditions of 37°C, and the medium was changed every 2 days.

1-9. In vitro Cellular Activities

The cell-containing sample was fixed in 2.5% glutaraldehyde for 4 hours, and the cell morphology was observed using the SEM according to Example 1-4. The samples were rinsed with 50%, 60%, 70%, 80%, 90% and 100% alcohol for 10 minutes, respectively. Hexamethyldisilazane was treated for 1 hour, and the sample was air dried. After coating with Au, the image was observed using the SEM according to Examples 1-4.

To evaluate the rate of cell proliferation, an MTT assay (Cell Proliferation Kit I, Boehringer Mannheim, Mannheim, Germany) was used. Samples were immersed in 0.6 mg mL ^-1 MTT solution at 37°C for 4 hours. The solubilization solution was then treated at 37° C. overnight. Using an ELISA reader (EL800, BioTek Instruments, Winooski, VT, USA), the absorbance was read at 570 nm after 1 and 7 days of incubation according to Examples 1-8. The MTT result on the 7th day and the MTT result on the 1st day were compared, and the relative cell proliferation rate was shown.

To measure cell viability at 4 hours (in situ) and 1 and 7 days, Live/dead staining was performed. The sample was immersed in 0.15 × 10 ^-3 m calcein AM and 2 × 10 ^-3 m ethidium homodimer-1 for 30 minutes. Then, green living cells and red dead cells were observed and captured under a microscope. From the images obtained, cell viability was calculated as the number of living cells relative to the total number of cells according to Examples 1-6.

DAPI / phalloidin images were obtained, and cell nuclei and F-actin were examined. Samples were fixed with 3.7% paraformaldehyde overnight at room temperature. After 10 minutes treatment with 0.2% Triton X-100, the sample was added to 5 x 10 ^-6 m DAPI (Invitrogen, Carlsbad, CA) and 15 U mL ^-1 phalloidin (Invitrogen, Carlsbad, CA) conjugated to Alexa Fluor 568. Soaked. Fluorescence images were captured using a confocal microscope (LSM700, Zeiss Blood Cell, Germany).

Immunofluorescence staining of MHC and sarcomeric α-actin structures was performed to indicate myoblast differentiation. After soaking 3.7% paraformaldehyde overnight at room temperature, the samples were permeated for 10 minutes using 0.2% Triton X-100. In order to block background noise, 1% bovine serum albumin (Invitrogen, Carlsbad, CA, USA) was treated for 1 hour. To attach the primary antibody, the sample was incubated overnight at 4°C with an anti-MHC antibody (1:200 in Tris buffered saline (TBS)) and an anti-sarcomeric α-actinin antibody (1:200 in TBS). . By counter staining with DAPI, samples were incubated with a goat antimouse secondary antibody conjugated with Alexa Fluor 488 for MHC and Alexa Fluor 568 for sarcomeric structures. Images were obtained using a confocal microscope, and ImageJ software according to Examples 1-4 was used for MHC analysis.

1-10. Statistical analysis

Statistical analysis was performed at least 3 times of each experiment using SPSS software (SPSS, Inc., Chicago, IL, USA). By single factor analysis of variance, p * <0.05, p ** <0.01 and p *** <0.001 were used to indicate statistical significance.

Example 2. Cell electrospinning for cell structure fabrication eletrospinning ; CE) process and polymer mixed solution Molecular entanglement (entanglement) check

The CE process was compared to the cell printing (CP) process using a 3D bio printer. 1A, according to Examples 1-4 showing a schematic diagram of a cell printing process used to prepare 3D cell-containing alginate struts according to Examples 1-5 and printed cell-containing struts. An SEM image is shown, and as shown in the bottom of FIG. 1A, a schematic diagram of CE and an SEM image of electrospun alginate micro/nano fibers containing cells are shown. In detail, the anisotropic array fibers were deposited on the parallel electrodes under an electric field applied between the nozzle and the parallel electrodes. The electrostatic motion of this fiber is due to the electric field induced torque, which can be expressed as T = μ×E. Where T is the field-induced torque, μ is the induced polarization moment, and E is the applied electric field.

In order to accurately observe the electric field distribution around the nozzle and the grounded electrode, the geometrical dimension of the electrospinning process indicated in the optical image was used, as shown in FIG. 1B. As can be seen from the simulation results of FIG. 1B, the electrospun fiber experiences a torque induced by an electric field by an induced dipole moment due to the electric field direction and shape factor of the parallel electrodes. In particular, as shown in Figs. 1b-I and 1b-II, the torque formula μ×E can be extended to 0.5Vε ₀ K _T (ω)E ² sin 2θ, where V is the volume of the fiber and ε ₀ is the free The permittivity of space, K _T (ε) is the difference between the depolarization factors for the two vertical axes of the fiber, ω is the applied frequency, E is the electric field, and θ is the rotation angle. The direction of rotation of the cylindrical structure may be directly affected by the direction of the electric field, as shown in FIGS. 1b-I and 1b-II. Based on this theoretical analysis, it was confirmed that electrospun fibers can be successfully aligned between parallel electrodes.

As shown in the optical image of FIG. 1B, the distance between the nozzle and the electric electrode of 140 mm and the distance between the electric electrode of 30 mm were fixed. Using a geometrical experimental setup, an attempt was made to prepare nanofibers using 2% by weight alginate, but no fibers were obtained (Fig. 1C). Pure alginate has a limit of electrospinning because the molecular chain is not entangled, but overlaps each other due to the strong and extended chain shape (Fig. 1c-III). Therefore, no matter how concentrated the domains of the alginate molecular chains are, as shown in Fig. 1c, entanglement of the molecular chains and fiber formation requiring a physical network cannot be achieved. In order to increase the low electrospinnability of alginate, polyethylene oxide (PEO) was used as a processing agent. PEO, as a carrier polymer, reduces surface tension and electrical conductivity, and more importantly, molecular entanglement required for electrospinning can be formed. As shown in Figure 1c, shows electrospun alginate/PEO fibers using alginate mixed with PEO (3% by weight). As expected, as shown in Figs. 1c-IV, it was confirmed that the alginate fibers aligned between parallel electrodes were well obtained due to the PEO effectively forming a physical network around the alginate molecular chain.

Example 3. Various PEO Fraction Having Alginate Electrospinning of the contained cell-containing bioink ( electrospinnability ) And Fiber Alignment to determine the optimal weight ratio

In order to determine the electrospinnable alginate/PEO solution (biocompatible polymer mixed solution), 2% by weight alginate was used as A2P0, A2P1, A2P2, A2P3 and A2P4 according to Examples 1-1 and 1-2. Each added with various weight fractions (0, 1, 2, 3 and 4% by weight) of the PEO mentioned respectively. As shown in FIG. 2A, changes in properties, surface tension, electrical conductivity, and complex viscosity of the mixture for various PEO fractions according to Example 1-3 are shown. An important factor that hinders the electrospinning of alginate is the repulsive force between the poly anions. As such, the addition of PEO to the alginate reduces the repulsion between the polyanionic alginate molecules, as evidenced by the change in conductivity, which indicates a decrease in the degree of ionization of the carboxylic acid salt. Further, molecular chain entanglement improves as the degree of ionization of the carboxylic acid salt decreases. Therefore, as shown in FIG. 2A, as the weight fraction of PEO in alginate increases, the decrease in surface tension, decrease in conductivity, and increase in viscosity are all advantageous for forming beads-free fibers. On the other hand, low conductivity and high viscosity increase the fiber diameter corresponding to the increase in fiber diameter from A2P2 to A2P4.

In addition, the alignment of the electrospun A2P1, A2P2, A2P3 and A2P4 between parallel electrodes was described in the SEM image according to Example 1-4, and as shown in FIG. 2B, the fiber distribution was expressed in grayscale, and A2P1 was fibrous. Did not form a structure. Therefore, the gray scale peaks were randomly distributed. However, A2P2 formed micro/nano fibers, but alignment between the electrodes was not achieved. On the other hand, A2P3 and A2P4 exhibited a flat fiber structure with a fully aligned structure. Quantitatively speaking, the half width (FWHM) was 37.0°, 8.4° and 12.0° in A2P2, A2P3 and A2P4, respectively, with A2P3 showing the lowest value indicating better alignment. Based on the electrospinability and alignment of the fibers, A2P3 prepared according to Examples 1-1 and 1-2 was used to study the subsequent parameters of CE for bead-free fiber formation and reasonably aligned fiber bundles. Was chosen.

Example 4. Confirmation of optimal processing conditions to generate aligned micro/nano fibers

Using a composition of alginate and PEO (A2P3), fiber formation was observed with respect to the nozzle-to-electrode distance and electric field. 3A shows a process window and a structure consisting of beads and beads and fibers, and fibers are represented by crosses (X), triangles (Δ), and circles (○). In this experiment, the flow rate and electrospinning time were fixed at 0.25 mL h ^-1 and 3 min, respectively. In the electrospinning condition, when the nozzle-electrode distance was less than 10 mm and the electric field was less than 0.075 kVmm ^-1 , formation of stable alginate fibers was not achieved. Therefore, 10-14 mm nozzle-electrode spacing and 0.075-0.125 kV mm-1 electric field range as shown in Fig. 3b, as a result of showing the SEM image according to Example 1-4, 12 ~ 14 mm nozzle-electrode Stable fiber structures (no beads and fiber arrangement) were observed in the interspace and 0.075 kV electric field regions. Based on the processing diagram, a stable range for forming alginate fibers was found.

In addition to producing stable fibers, cell viability is an important factor in determining the success of CE. To observe cell viability over a fixed treatment range (14 mm distance and 0.050-0.125 kV mm ⁻¹ electric field), A2P3 was used in a mixture of C2C12 cells (5×10 ⁶ cells mL ⁻¹ ). 3C shows SEM and fluorescence images according to Examples 1-4 of electrospun alginate fibers containing cells. Green and red represent live and dead cells, respectively. High cell viability was observed, but as expected, when 0.05 kV mm ^-1 was used, a non-fibrous structure was formed. In the case of 0.075 kV mm ^-1 , formation of stably aligned alginate fibers and reasonable cell viability (90%) were achieved. However, as shown in FIG. 3D, at 0.1 kV mm ^-1 or more, the cell viability was significantly reduced to 80%, and the fibrous structure began to be largely condensed. When a high current was applied to the cells, a large hole was formed in the membrane due to rupture, eventually leading to death, and the lowest cell viability was observed at 0.125 kV mm ^-1 . Therefore, a 14 mm nozzle-electrode spacing and a 0.075 kV mm ^-1 electric field were set to obtain a fibrous bundle structure containing cells. Using the selected conditions, fiber bundles before and after crosslinking were evaluated in terms of structural stability, as shown in Fig. 3E. Fibers after crosslinking treatment in which PEO was dissolved showed some fusion areas, but individual fibrous topological cues and structural stability were obtained in an ordered manner.

After setting the A2P3 solution, the nozzle-electrode distance and the electric field, the effects of the electrode distance and the electrospinning time of the parallel electrode were evaluated to produce aligned fibers. The electrode-to-electrode distance was changed to 5 to 40 mm, as shown in FIGS. 4A and 4B, and the FWHM and the number of fibers were measured. Since the electrode is too close at a distance of 5 mm, the fiber deposition can become unstable. As shown in FIG. 4B, the FWHM exponentially decreases to a distance of 5 to 30 mm, and increases at 30 mm or more. At 40 mm, the deposited fibers broke easily; Therefore, they did not develop into ordered fiber bundles as shown in the optical image. In addition, the fiber density per 10 ⁴ μm ² was measured. The fiber density at a parallel electrode distance of 20 mm was the highest compared to other distances. This is because the distance can be the lowest point of the charge deposited by the electrospun fiber. However, since the alignment of the electrospun fibers is highest at 30 mm, the next distance was chosen.

As shown in Fig. 4c, fiber formation was evaluated with electrospinning times of 60, 180, 300 and 420 seconds using a 30 mm distance. As a result, as shown in FIG. 4D, the FWHM of the deposited fiber was lowest at 180 seconds, and by increasing it to more than 180 seconds, the FWHM increased significantly due to the residual charge of the deposited fiber. Therefore, 180 seconds was the best choice for the electrospinning time.

Example 5. Electrospun cells Cell support (Cell- Electrospun Scaffold; CE Cell support ) Fabrication and confirmation of cell activity in vitro

From the parametric study, cell eletrospinning (CE) was performed with a 14 mm nozzle-electrode distance, a 0.075 kV mm ^-1 electric field, a 30 mm electrode-to-electrode distance, and a 180 s electrospinning time. Subsequently, the CE process according to Examples 1-5 was compared with the cell printing (CP) process, as shown in FIG. 5A, respectively. The CP process has been studied in depth in order to observe the cell activity according to Examples 1-9 in tissue engineering, so the CP cell scaffold was set as a control. Specifically, the CP process was chosen to compare the effect of microtopological cues on cellular activity. To prepare the CP scaffold, the same bioink used for the CE cell scaffold, A2P3 containing C2C12 cells, and a 3D printer operated at 120 kPa air pressure and 10 mm s ⁻¹ nozzle movement speed were used. The CP and CE cell scaffolds were made of a single strut with a diameter of 70-100 μm and a length of 30 mm. In addition, the number of cells per area (mm ² ) according to Example 1-6 was measured, and for the CP cell support and the CE cell support, it was 1868 ± 190 and 1906 ± 265. The number of cells was similar between the two cell scaffolds, respectively. As shown in FIG. 5A, optical and SEM images according to Examples 1-4 show different surface morphologies between the CP cell scaffold and the CE cell scaffold. The CP cell scaffold showed a relatively smooth surface containing cells, but the CE cell scaffold showed arrayed microfibers containing cells. All the produced artificial cell scaffolds exhibited in situ cell viability of 90% or more in the survival/kill image of FIG. 5A, and the process was proved to be a biocompatible platform.

The characteristics of the CP cell support and the CE cell support are as shown in Figs. 5b to 5c. That is, in order to evaluate the toxicity before and after the crosslinking process using ₂ % by weight CaCl ₂ for 2 minutes, as shown in FIG. 5B, in situ cell viability was measured. Statistically, the crosslinking process does not affect cell viability for both cell scaffolds and pure bioink that contains cells but is not printed. This means that the manufacturing and crosslinking process is applicable to cells. In addition, the tensile test according to Examples 1-7 was performed using a uniaxial tensile machine, as shown in FIG. 5C. The stress-strain curves were obtained, and by the initial slope, the tensile modulus was measured as 5.1 ± 1.0 and 5.0 ± 2.9 MPa for the CP cell scaffold and CE cell scaffold, respectively. Although the tensile moduli were not significantly different, the CE cell scaffolds have a maximum tensile strength (at maximum tensile strength), as the individual microfibers fabricated using the CE process are stretched more efficiently due to the evenly distributed stress concentration. It was confirmed that the maximum tensile strength) and strain were higher.

In FIGS. 5D to 5G, in vitro cellular activity of the CP scaffold and CE scaffold were evaluated. Both cell scaffolds maintained a high cell viability of 90% or more for 1 and 7 days of culture according to Examples 1-8, and as shown in FIGS. 5D and 5E, this is a biocompatibility for increasing the culture time of the cell scaffold. Indicated that it is the environment. In addition, when comparing the cell proliferation rate on the 7th day with the cell proliferation rate on the 1st day, as shown in Fig. 5f, the results for the CP cell support and the CE cell support were 122% and 121%, respectively. After living cells successfully settled in the structure, they were able to proliferate and maintain metabolic functions. As a proof of concept, CE cell scaffolds were fabricated with small diameters (about 60 μm) to demonstrate several individual cells contained in the fibrous structure. As shown in Fig. 5G, the optical image shows the micro/nano fiber structure and the cells located along the alginate microfibers. The cells were alive in the / image, and F-actin was expressed around the nucleus of the diamidino-2-phenylindole (DAPI) / phalloidin image on day 1. In addition, the expression of MHC was also observed on day 7, but a longer cell culture period was required for cell-to-cell interaction. Without physical support, fibrous structures made using the CE process can easily bend or deform due to their low mechanical stability. Therefore, a mechanical improvement is needed to obtain a successful implant.

Example 6. hybrid Mechanical reinforcement of cell-containing fiber bundles using structure

In order to increase the mechanical stability, the CP cell support and the CE cell support were supplemented with a PCL brace of 300 μm, as shown in FIG. 6A. As can be seen from the schematic image, a bio-ink containing cells and a cylindrical PCL rod covered with fine fibers containing cells were applied to the CP and CE processes according to Examples 1-5, respectively. As shown in FIG. 6B, the shape of the structure according to the presence/absence of the PCL brace was demonstrated by the optical image. Without PCL braces, the CP and CE cell scaffolds are very flexible and can be easily bent when handled with forceps. When the PCL brace was used as a support structure, the cell support retained its original shape even when handled with forceps. In addition, the tensile properties according to Examples 1-7 of the PCL brace were compared with the printed and electrospun alginate, as shown in FIG. 6C. The modulus of PCL is 107.1 ± 12.8 MPa, which is far superior to CP and CE cell scaffolds without PCL braces. 6D shows a cross-section of a DAPI/phalloidin image of a cell scaffold using a PCL brace. On day 1, cells were evenly distributed on the CP and CE cell scaffold surfaces. Subsequently, the cells proliferated for 7 days, and the cytoskeleton of the cells on both cell scaffolds was enlarged and completely covered the surface of the PCL, as evidenced by the schematic diagram of FIG. 6A. In addition, to form a muscle-mimicking bundle structure, as shown in Fig. 6e, dozens of cell-containing PCL braces can be stacked. By this method, the muscle-mimicking bundle structure can be formed to a macroscopic size, which can be a more realistic structure for clinical application.

Example 7. Confirmation of In vitro Cellular Activities including Differentiation

Cellular activity according to Examples 1-9 was investigated using CP and CE cell scaffolds supplemented with PCL braces. As shown in Figures 7a and 7b, the cell viability was obtained from the cell scaffold without PCL, that is, corresponding to 90% or more for both CP and CE cell scaffolds for 7 days of culture according to Examples 1-8. This shows that the CP and CE processes according to Examples 1-5 using PCL braces are safe and biocompatible for the contained cells. In addition, cell morphology was captured by SEM according to Examples 1-4, as shown in FIG. 7C. On the 7th day, cells on the CP cell scaffold were attached in a circle on the surface of the PCL brace. On the other hand, cells on the CE cell support were anisotropically elongated along the direction of the micro/nano fibers. In addition, as shown in FIG. 7D, the DAPI/phalloidin image shows the same characteristics of round cells on the CP cell support and the uniaxially stretched cells on the CE cell support. For quantitative analysis, DAPI/phalloidin images were measured with the aspect ratio and orientation of F-actin. When the CE cell scaffold and the CP cell scaffold were used, the cell behavior in the 2D culture plate was completely different, so the cell morphology in the 3D environment was compared with or without a fine pattern. As shown in Fig. 7e, the F-actin aspect ratio indicating cell elongation was 2.8 times higher in CE cell scaffold than CP cell scaffold. In addition, F-actin orientation was indicated by FWHM, and lower values indicated better cell sorting. The FWHM of the CE cell scaffold was 3.0 times lower than that of the CP cell scaffold, because FWHM was able to grow on fibers aligned at 12°. In other words, the CE cell scaffold is more advantageous for cell elongation and alignment by inducing aligned on the scaffold surface.

The degree of myogenic differentiation was evaluated by myosin heavy chain (MHC) and sarcomeric α-actin staining. Since it was not possible to provide a topological signal to the cells containing the CP cell scaffold, it was used as a negative control to observe the myoblast differentiation to the CE cell scaffold. As shown in Fig. 7f, MHC, a protein required for myotube formation, was expressed to indicate myogenesis on days 7, 14, and 21. CP cell scaffolds showed MHC expression in multipolar morphology over 21 days, whereas MHCs on CE cell scaffolds were aligned and joined in a uniaxial direction. 7G shows the MHC positive index showing the ratio of MHC-positive nuclei to the total number of nuclei. The MHC positive index of the CE cell scaffold reached about 90% at 7 days, but the value of the CP cell scaffold was only 34%. As shown in Fig. 7h, among the MHC-positive nuclei, MHC regions having two or more nuclei were integrated to indicate the fusion index. The fusion index of the CE cell scaffold was 1.6 times higher than that of the CP cell scaffold on day 14, which was more than 90%, indicating that MHC-positive cells on the CE cell scaffold began to develop into a fusion structure. The maturation index was calculated by the number of MHCs having 5 or more nuclei and the number of MHCs having 2 or more nuclei, as shown in FIG. 7i. On the 21st day, the CE cell scaffold showed a significantly higher maturity index than the CP cell scaffold, and reached 70% or more. From the above results, cell fusion and development to the mature differentiation stage was effectively achieved by the ordered fibrous structure. In order to confirm the mature differentiation stage of the myotube, a myotube (sarcomeric) structure was expressed on the 21st day. This is because it can only be found in multinucleated myotubes that are precisely aligned with α-actin and Z line. As shown in Fig. 7J, the optical image proves the cultured sample transferred with tweezers after 21 days of culture according to Examples 1-8. Fluorescent images show an ordered multinucleated sarcomeric structure that is remarkably developed in the CE cell scaffold. These results confirmed that the fibrous structure on the CE cell scaffold induces cell-cell interaction, root canal formation, and fusion in a unidirectional array.

The above description of the present invention is for illustrative purposes only, and those of ordinary skill in the art to which the present invention pertains will be able to understand that it is possible to easily transform into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative and non-limiting in all respects.

Claims (15)

A method for producing a cell scaffold for regeneration of skeletal muscle tissue having a fibrous structure arranged in one direction comprising the following steps:
(a) preparing a biocompatible polymer mixed solution by mixing alginate and polyethylene oxide (PEO);
(b) preparing a myoblast-containing bioink by mixing myoblasts with the biocompatible polymer mixed solution prepared in step (a); And
(c) The myoblast-containing bioink prepared in step (b) is used as parallel electrodes with an electrode-electrode spacing of 5 to 30 mm under the condition of an electric field strength of 0.050 to 0.100 kV/mm for 60 to 300 seconds. During electrospinning of cells (cell eletrospinning; CE), a cell scaffold having a myoblast-containing nanofiber bundle structure in which the myoblasts are arranged in a living state is prepared. The method of claim 1,
In the step (a), the biocompatible polymer mixed solution is characterized in that the weight ratio of alginate/PEO is 0.5 to 2, the method for producing a cell scaffold for skeletal muscle tissue regeneration. The method of claim 2,
The biocompatible polymer mixed solution has a surface tension of 38 to 48 mN/m, an electrical conductivity of 2.9 to 3.9 μS/cm, or a complex viscosity according to the weight ratio of alginate/PEO. 1 ~ 15 Pa · S, characterized in that, the method for producing a cell support for skeletal muscle tissue regeneration. delete delete delete A cell support for regeneration of skeletal muscle tissue having a fibrous structure arranged in one direction prepared by the method of any one of claims 1 to 3,
The cell scaffold for skeletal muscle tissue regeneration is characterized in that it has a myoblast-containing nanofiber bundle structure in which myocytes are arranged in a living state. The method of claim 7,
The arrangement of the myoblast-containing nanofiber bundle structure is characterized in that the full width at half maximum (FWHM) is 10 to 40 °, the skeletal muscle tissue regeneration cell scaffold. The method of claim 8,
The myoblasts have an aspect ratio of F-actin of 4 to 6, or an orientation of F-actin of FWHM of 0 to 20°, characterized in that, skeletal muscle tissue regeneration cell scaffold. The method of claim 7,
The cell scaffold for skeletal muscle tissue regeneration is characterized in that the myosin heavy chain (MHC) is expressed, a cell scaffold for skeletal muscle tissue regeneration. The method of claim 10,
The MHC is characterized in that the MHC positive index (MHC positive index) is 90 to 100%, the fusion index (Fusion index) is 30 to 90%, or the maturation index (Maturation index) is 30 to 70%, skeletal muscle tissue regeneration Cell support. The method of claim 7,
The cell scaffold for skeletal muscle tissue regeneration is characterized in that the sarcomeric structure is expressed, the cell scaffold for skeletal muscle tissue regeneration. The method of claim 7,
The cell scaffold for skeletal muscle tissue regeneration has a cell viability of 80 to 95%, a FWHM of 10 to 40°, a fiber density of 120 to 240/μm ² , or a tensile strength of 30 to 35 MPa, or a relative cell proliferation rate ( Relative cell proliferation) is 100% ~ 122%, characterized in that, skeletal muscle tissue regeneration cell support.
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