KR102170467B1 - Cell scaffold with fiber bundle structure and method of manufacturing the same - Google Patents

Abstract

The present invention relates to a cell scaffold having a fibrous bundle structure and a method for manufacturing the same, wherein the cell scaffold manufacturing method of the present invention prepares a structure by mixing PVA and a polymer, and removes PVA, so that cells having a surface structure similar to that of muscle tissue in the body A scaffold can be prepared, and a cell scaffold having a fibrous bundle structure provides a three-dimensional microenvironment optimized for growth and maturation of muscle cells and differentiation of myoblasts into muscle cells. Therefore, it is expected that the cell support of the present invention can provide a muscle cell culture substrate skeleton for regeneration of muscle tissue.

Description

Cell scaffold with fiber bundle structure and method of manufacturing the same}

The present invention relates to a cell support having a fiber bundle structure and a method for preparing the same, and more specifically, a cell support having a fiber bundle structure prepared by a method of removing PVA after preparing a strut using a mixture of a biocompatible polymer and PVA. Etc.

Muscles are essential tissues that support our body and maintain life phenomena. In the muscle, myoblasts differentiate to form multinucleated myotubes and create myocytes. Musculoskeletal tissues, including muscles, ligaments, and tendons, have various three-dimensional arrangements or patterns. Cells can detect topographical signals that affect cell behavior and control a variety of factors, including cell type, size, and shape. In order to restore the functional properties of tissues, a biomimetic scaffold that mimics the microenvironment of tissues is used, and according to the recent development of micro-/nano-technology, a two-dimensional micro-/nano-topology The structure was developed.

On the other hand, the skeletal muscle tissue that performs the function of contracting in the direction in which the fiber bundle extends because it is composed of very densely aligned fiber bundles is a phase structure of muscle tissue consisting of muscle fibers sequentially formed by a combination of single muscle cells. And the alignment of muscle cells forms myotubes, which is considered to affect muscle development. In other words, it means that the alignment of muscle cells is very important for the regeneration of muscle tissue.

Nanofibers using electrospun technology can induce alignment of muscle cells, and it was confirmed that electrospun chitosan-PCL [PCL, poly (ε-caprolactone)] nanofibers induce cell alignment and form myotubes (Jana , S.; Leung, M.; Chang, J.; Zhang, M. Effect of nano- and micro-scale topological features on alignment of muscle cells and commitment of myogenic differentiation.Biofabrication 2014, 6 (3), 035012.) . In addition, it has been found that when cells are cultured on a polystyrene substrate imprinted with an intaglio using a master mold manufactured through photolithography as a template, it is possible to align muscle cells. However, cell culture on the substrate produced by the imprint process has a drawback that although it affects the alignment of muscle cells, it does not significantly affect the differentiation of myoblasts into muscle cells.

Another way to align muscle cells is to culture the cells on a micropatterned substrate with cell adhesion proteins. This is to provide topographic characteristics that affect the differentiation into muscle cells and the formation of myotube cells by attaching fibronectin, laminin, or collagen to the cell culture dish using a microcontact printing technique.

However, the previously studied pattern formation method only provides a two-dimensional (2D) surface structure, and the 2D surface structure allows the alignment of myoblasts, but there is a problem in that it is not efficient for the formation of myotubes and myofibers. In order to overcome the limitation of the 2D surface structure, various studies are being conducted to develop a three-dimensional (3D) surface structure.

The technical task to be achieved by the present invention is ultimately for regeneration of muscle tissue, and a three-dimensional structure of a cell support having a fine pattern to form myotubes by inducing uniaxial alignment of muscle cells during muscle cell culture. To provide.

Accordingly, it is an object of the present invention to provide a method for producing a cell scaffold having a fibrous structure similar to that of muscle tissue and a cell support prepared by the method.

In addition. Another object of the present invention is to provide a method for promoting differentiation of myoblasts into muscle cells, including culturing myoblasts on the cell support, and a cell therapy product comprising muscle cells differentiated by the method.

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 will be clearly understood by those skilled in the art from the following description.

In order to solve the above technical problem, the present invention comprises the steps of: (a) preparing a PVA/polymer mixture by adding a biocompatible polymer to an amorphous polyvinyl alcohol (PVA) solution; (b) extruding the PVA/polymer mixture to uniaxially arrange it; (c) lyophilizing the arranged PVA/polymer mixture; And (d) it provides a method for producing a cell scaffold having a fiber bundle structure comprising the step of removing the PVA by washing the PVA/polymer mixture.

In another embodiment of the present invention, in the step (a), the PVA/polymer mixture is PVA It may be mixed in a ratio of 1 to 5: 5 to 9.

In another embodiment of the present invention, the flow rate of the PVA/polymer mixture during extrusion in step (b) may be 0.01 to 0.2 μL/s, and preferably 0.02 to 0.08 μL/s.

In another embodiment of the present invention, the extrusion of step (b) may be rotational extrusion.

In yet another embodiment of the present invention, the manufacturing method comprises adding an EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) solution to the lyophilized PVA/polymer mixture after step (c) to obtain the polymer. It may further comprise the step of crosslinking.

In another embodiment of the present invention, the crosslinking step may be performed at room temperature for 10 to 50 minutes, preferably 20 to 40 minutes, more preferably for about 30 minutes.

In another embodiment of the present invention, the biocompatible polymer may be a synthetic polymer or a natural polymer.

In another embodiment of the present invention, the synthetic polymer is poly-ε-caprolactone (PCL), poly-lactic-co-glycolic acid (poly(lactic-co-glycolic acid): PLGA), and polylactic acid. It may be one or more polymers selected from the group consisting of (polylactic acid: PLLA).

In another embodiment of the present invention, the natural polymer may be one or more polymers selected from the group consisting of collagen, chitosan, alginate, and gelatin.

In addition, the present invention provides a cell support having a fiber bundle structure by removing polyvinyl alcohol from a structure prepared by mixing amorphous polyvinyl alcohol (PVA) and a biocompatible polymer.

In addition, the present invention provides a method for inducing differentiation or promoting differentiation of myoblasts into muscle cells, comprising culturing myoblasts on the cell support.

In one embodiment of the present invention, the muscle cells may be skeletal muscle cells.

According to the method for producing a cell scaffold of the present invention, a structure is prepared by mixing PVA and a polymer, and PVA is removed to prepare a cell scaffold having a surface structure similar to that of the muscle tissue in the body composed of a bundle of muscle fibers. In the case of culturing muscle cells in the cell scaffold of the present invention, the proliferation is active and bipolar extension is similar to that of muscle cells, and root canal formation is promoted to enable culture into muscle fibers. In addition, when culturing myoblasts on the cell scaffold of the present invention, the differentiation into muscle cells is promoted. The cell scaffold having a fibrous bundle structure of the present invention includes growth and maturation of muscle cells and muscle cells of myoblasts. By providing a three-dimensional microenvironment that is optimized for differentiation into a furnace, it is expected to be able to provide a skeleton for a muscle cell culture substrate for muscle tissue regeneration. In addition, the cell support of the present invention is expected to be usefully used not only for muscle regeneration, but also for tissue regeneration consisting of an array of structures such as spinal cord, nerve tissue, and ligaments.

1A is a schematic diagram of a step of preparing a fibrous PVA solution by applying a high temperature to a semi-crystalline PVA to transfer a phase to an amorphous PVA.
Figure 1b is a PVA/collagen mixture extruded through a nozzle to uniaxially arrange, freeze-dry, and add EDC to induce crosslinking of collagen, and wash with distilled water to remove PVA to prepare a collagen cell support having a fiber structure. It is a schematic diagram of how to do it.
2 is a scanning electron microscope (SEM) image (a to d) of the surface of a cell support prepared by varying the PVA content, and topology characteristics (e), storage modulus (f), and complex viscosity of the cell support according to the PVA content ( This is a graph comparing the difference between g).
3A is a scanning electron microscope image of a collagen strut prepared with pure collagen and a collagen strut prepared with a PVA/collagen mixture (PVA: collagen=1:1).
3B is a Fourier showing distribution of orientation (d) and PVA components of collagen struts prepared with pure collagen and collagen struts prepared with PVA/collagen mixture (PVA: collagen=1:1) and PVA components completely leached from PVA/collagen struts. This is a graph comparing the converted infrared (FT-IR) result (e).
4A is a scanning electron microscope image of the surface of a collagen strut prepared with a mixture of collagen and PVA in different ratios.
FIG. 4B is a graph comparing the distribution of orientation (e) and rheological properties (f) of collagen struts prepared with a mixture of different ratios of collagen and PVA.
Figure 4c shows the difference between the distribution of orientation (k) and diameter (l) of the strut according to the flow rate and the scanning electron microscope image (g to j) of the strut surface prepared by varying the flow rate when extruding the PVA/polymer mixture. This is a comparison graph.
5 is an image of the surface and cross-section of the PCL strut prepared by varying the content ratio of PCL and PVA ((d) pure PCL, (e) PCL: PVA = 7:3, (f) PCL: PVA = 3:7. ), a graph comparing the difference in distribution of orientation (c) according to the content ratio, a schematic diagram of a printing method for rotationally extruding a PVA/polymer mixture (h), and an image of the PCL structure manufactured by the rotational extrusion printing method (i And j).
6 is a graph comparing the distribution of orientation (c) with optical and scanning electron microscope images of CCS (a) and CSAT (b) cell scaffolds.
Figure 7a shows images (a and b) obtained by fluorescence staining of live cells (green) and dead cells (red) after 1 and 3 days of culturing cells on a CCS or CSAT cell support, and MTT assay analysis. It is a graph (c) evaluating cell proliferation after 1, 3, and 7 days have elapsed.
7B is an image (d and e) obtained by fluorescence staining of nuclei (blue) and F-actin (red) after 7 and 14 days of culturing cells on a CCS or CSAT cell support, and after 14 days. This is a graph comparing the difference in distribution of orientation(f).
Figure 8a is an image obtained by culturing cells on a CCS or CSAT cell support, and fluorescently staining the nuclei (blue) and MHC (green) after 7, 14, and 21 days have elapsed.
8B is a graph comparing the difference in distribution of orientation after 7 and 14 days of culturing cells on a CCS or CSAT cell support.
Figure 8c is a graph of culturing cells on CCS or CSAT cell support and analyzing myosin heavy chain (MHC) ((f) MHC positive area, (g) myotube length, (h) myotube diameter, (i) fusion index, and (j) maturation index) is a graph comparing the expression levels of Myod1, Myf5, myogenin (Myog), and Myh2. Gene expression was compared with β-actin and normalized to the value of cells cultured on the CCS cell support.

The present inventors focused on the important influence of the fine phase pattern in cell culture, and as a result of intensive research to develop a three-dimensional fine pattern structure optimized for differentiation and cultivation of muscle cells, PVA was used in muscle tissue. It was confirmed that a fibrous structure that is uniaxially arranged as a bundle of muscle fibers can be produced, actively proliferates when culturing muscle cells from the produced structure, forms a myotube, and forms the myoblast's myocyte. ) By confirming that the differentiation proceeds rapidly to complete the present invention.

More specifically, the present inventors applied high temperature or high pressure to crystalline or semi-crystallin polyvinyl-achohlo (PVA) to form a fibrous PVA by phase transition to PVA of an amorphous structure. It was obtained, mixed with a polymer, and extruded through a printing process to prepare a PVA/polymer structure. Subsequently, the prepared PVA/polymer structure was washed with distilled water to remove PVA, thereby obtaining a cell scaffold having a fiber bundle surface structure optimized for culturing or differentiation of muscle cells.

In a specific embodiment of the present invention, a collagen structure produced through a printing process, a lyophilization process, a crosslinking process, and a PVA removal process by mixing PVA and collagen, a pure collagen structure, and a PVA/collagen structure in which PVA is not removed The surface of is confirmed through a scanning electron microscope and the degree of alignment was quantified by FWHM (full width at half maximum). As a result, the surface of the pure collagen structure and the PVA/collagen structure from which PVA was not removed has a smoother surface, whereas After the PVA was removed, in the case of the collagen structure, a uniaxially arranged micro pattern similar to the surface of the fiber bundle was formed, and the degree of alignment (FWHM) was also considerably high at 14.7° (see Example 2-2 and FIG. 3A).

Accordingly, the present invention comprises the steps of: (a) preparing a PVA/polymer mixture by adding a biocompatible polymer to an amorphous polyvinyl alcohol (PVA) solution; (b) extruding the PVA/polymer mixture to produce a single uniaxially arranged strut; (c) lyophilizing the arranged PVA/polymer struts; And (d) washing the PVA/polymer strut to remove the PVA, to provide a method for producing a cell scaffold having a fiber bundle surface structure.

On the other hand, so that the binding between polymers can be maintained even after PVA is removed, the present invention provides an EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) solution to the lyophilized PVA/polymer strut after step (c). It may further include the step of crosslinking the polymer by adding.

In addition, the extrusion of the PVA/polymer mixture in step (b) may be performed using a 3D printer, and at this time, a nozzle or a nozzle tip may be rotationally extruded.

In a specific embodiment of the present invention, the optimal conditions for formation of the microsurface structure in the manufacture of a cell culture scaffold having an arranged three-dimensional fiber bundle structure were analyzed in detail. More specifically, by varying the content of PVA and the mixing ratio of PVA and polymer in step (a) and the pressure when extruding the PVA/polymer mixture in step (b), that is, the conditions of flow rate, respectively, to prepare a cell support, By observing the surface, a cell scaffold having a more muscle tissue-like surface was identified to determine the optimum conditions (see Examples 2 and 3).

The present inventors prepared a cell scaffold by varying the content of PVA to 5, 10, 15, and 20% by weight in a PVA/collagen mixture in order to confirm the optimal PVA content for fabricating the surface structure of a uniaxially arranged fiber bundle, and the surface As a result of confirming, when the content of PVA was 15-20% by weight, it was confirmed that the fiber bundle structure of the collagen structure was clearly displayed after the PVA was removed (see Example 2-1).

Therefore, the weight percent of the PVA is not limited as long as the surface of the cell culture support obtained after PVA removal has a fiber bundle pattern, but when too little PVA is included, a block-shaped pattern is formed after PVA removal. If too much PVA is included, there is a problem in that the PVA viscosity is too high to facilitate binding to collagen. Therefore, in the preparation method, the PVA/polymer mixture of step (a) may contain 12 to 25 wt% of PVA, preferably 15 to 22 wt%, more preferably 20 (±1) wt% can do.

In addition, the present inventors mixed collagen and PVA at 7:3, 5:5, 3:7, and 1:9, respectively, in order to confirm the optimal PVA/polymer mixing ratio for fabricating the surface structure of uniaxially arranged fiber bundles. As a result of preparing a cell scaffold using a collagen/PVA mixture and analyzing its surface, in the case of a ratio of 7:3, a uniaxial phase pattern could not be observed, but in the case of other ratios, a uniaxially arranged fiber bundle was formed. The phase pattern could be observed. On the other hand, in the case of the ratio of 1:9, it was possible to confirm the uniaxially arranged phase pattern, but the structure of the unstable strut was shown after PVA leaching (see Example 2-3).

Therefore, the polymer/PVA mixing ratio is not limited as long as the surface of the cell culture support obtained after PVA removal has a fiber bundle pattern, but may be a ratio of 1 to 5: 5 to 9. That is, PVA may be included in an amount of 0.5 to 9 parts by weight, preferably 1 to 9 parts by weight, and more preferably 1 to 2.3 parts by weight, based on 1 part by weight of the polymer.

In addition, the present inventors prepared a cell scaffold by varying the flow rate of the mixture to 0.02-0.11 μL/s during extrusion in order to confirm the optimum flow rate during extrusion for the fabrication of the surface structure of a uniaxially arranged fiber bundle, and analyzed the surface. , When the flow rate was low, an unstable fiber pattern was indicated, and when the flow rate was high, it was confirmed that the diameter of each pattern arranged uniaxially increased (see Example 2-4).

Therefore, the flow rate of the mixture during extrusion is not limited as long as the surface of the cell culture support obtained after PVA removal has a fiber bundle-like pattern, but is preferably 0.01 to 0.2 μL/s, preferably 0.02 to 0.11 μL/s, more preferably 0.08 (±0.02) μL/s.

When the content of PVA was 15-20% by weight, it could be confirmed that the fiber bundle structure of the collagen structure was clearly displayed after PVA was removed (see Example 2-1).

Meanwhile, in the present invention, the'polymer' is a water-soluble biocompatibie polymer that forms the skeleton of a cell culture substrate, and includes synthetic polymers and natural polymers. The biocompatible polymer refers to a polymer having a characteristic that does not exhibit rejection when contacted with biological tissues, cells obtained therefrom, and body fluids.

In the present invention, the term'synthetic polymer' is an organic chemical engineering and is not limited as long as it is a water-soluble polymer, and non-limiting examples thereof include Polycaprolactone (PCL), Poly(lactic-co-glycolic acid) (PLGA), and Polylatic acid. (PLLA), etc.

In addition, in the present invention, the'natural polymer' may be collagen, cellulose, starch, chitosan, alginate, and gelatin.

On the other hand, in a specific embodiment of the present invention, polyvinyl alcohol was mixed with amorphous PVA, an extrusion process, and a PVA removal process were performed to prepare a PCL cell support, and the surface was analyzed. As a result, a micropatterned surface in the form of a fiber bundle It was confirmed that, in particular, when the nozzle is rotated during extrusion, it was confirmed that a helical cell support having a fine pattern in the form of a fiber bundle can be obtained (see Examples 1-5 and Example 3).

In addition, in a specific embodiment of the present invention, as a result of culturing myoblasts on the cell support prepared by the above method and observing the degree of cell survival through MTT assay and fluorescent staining, the cells survive at a high level in the cell support. It was confirmed that it can proliferate. In particular, from the results of fluorescence staining of the nucleus and cytoskeleton (F-actin), it was found that when cultured on a cell support having a uniaxially arranged pattern structure of the present application, it proliferates in the form of bipolar kidneys aligned in parallel similar to the cell elongation in muscle tissue. Confirmed. In addition, from the results of analyzing the expression of the muscle cell marker gene, it was confirmed that the cell scaffold provided by the present invention promotes the differentiation of myoblast into muscle cells, and the muscle tissue in the body is composed of multinuclear muscle fibers. As in the case, several nuclei were identified in the MHC-positive region, and it was found that it is advantageous for myotube formation (Example 4).

From the above, the cell scaffold provided by the present invention can be used as a cell culture substrate for promoting differentiation of myoblasts into muscle cells, and the muscle cells cultured in the cell scaffold of the present invention have active metabolic activity, The muscle cells obtained by culturing on the cell support of the present application can be used as a cell therapy agent that can be administered to an individual in need of prevention or treatment of muscle diseases.

On the other hand, the cell support of the present invention is composed of a biocompatible polymer and can be used as a bio-insertion device for regeneration of muscle tissue. When used as a biological device, the cell support is preferably made of a biodegradable polymer.

Since the present invention can apply various transformations and have various embodiments, specific embodiments are illustrated in the drawings and will be described in detail in the detailed description. However, this is not intended to limit the present invention to a specific embodiment, it is to be understood to include all conversions, equivalents, and substitutes included in the spirit and scope of the present invention. In describing the present invention, when it is determined that a detailed description of a related known technology may obscure the subject matter of the present invention, a detailed description thereof will be omitted.

Hereinafter, a preferred embodiment is presented to aid the understanding of 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 1. Experimental method and material

1-1. Materials

PVA (density = 1.27 g/cm ³ , Mn = 89000-98000 g mol ^-1 ) and PCL (Mn = 45000 g mol ^-1 , T _m = 60 °C) were obtained from Sigma-Aldrich Co. (St. Louis, USA). Type I collagen 4% (MatrixPSP, purity: 98% or higher, Bioland, Cheonan-city, South Korea) was obtained from pig skin. EDC (Mw = 191.7) solution for crosslinking collagen was purchased from Sigma-Aldrich and used.

1-2. In situ fibrosis method of PVA

A PVA fibrosis process was performed to prepare a collagen strut having a uniaxially aligned surface structure. More specifically, PVA was dissolved in distilled water for 10 minutes in an autoclave at 120° C. to form fibers. According to previously conducted studies, the hydroxyl groups of PVA combine with water, and the structure of the hydrogel is microfibrils through wall shear stress that weakens the bonding force between water and PVA in the 3D printing nozzle. microfibrill) form.

1-3. Method for producing a collagen structure with a fine pattern

PVA was dissolved in distilled water for 10 minutes in an autoclave at 120° C. to form fibers, and 4 wt% collagen was mixed with 5, 10, 15 or 20 wt% PVA solution. When amorphous PVA and collagen are mixed, collagen is mixed between the hydroxyl groups of PVA. A low-temperature plate (-10 ℃) was used to fabricate the cell scaffold, and a three-axis printing system (DTR3-2210 T-SG, DASA Robot, Seoul, South) with a moving speed of 20mm/s of the nozzle extruding the PVA/polymer mixture. Korea) was used. A 30G nozzle having an inner diameter of 150 μm was used to manufacture PVA/collagen struts, and the flow rate of the mixture was adjusted to 0.08 μL/s during extrusion. PVA/collagen struts were freeze-dried at -76°C for 12 hours using an SFDSM06 freeze dryer (Samwon, Busan, South Korea), and then 50mM EDC solution was added together with 95% ethanol for 24 hours at room temperature. Collagen crosslinks were formed. Subsequently, by washing three times with distilled water at 37° C. for 4 hours, PVA/collagen struts were removed from PVA. The collagen structure from which PVA was removed was freeze-dried again at -76°C for 12 hours.

1-4. Method of manufacturing PCL structure with fine pattern formed

In order to prepare a synthetic polymer structure in which a fine pattern is topologically formed, 20% by weight of PVA is dissolved in distilled water for 10 minutes in an autoclave at 120° C., and various% by weight of PCL is added to the PVA solution to prepare a PVA/PCL solution. Was prepared. Then, water in the PVA/PCL solution was evaporated at 120° C. to obtain a PVA/PCA mixture. The moving speed of the nozzle for extruding the mixture was adjusted to 20mm/s, and a 22G nozzle was used as the nozzle. The heating barrel temperature of the printer was set to 85° C., and the air pressure was set to 250 kPa. After preparing the PCL/PVA structure, PVA was removed by washing three times with distilled water at 37° C. for 4 hours, and the PCL structure from which PVA was removed was freeze-dried again at -76° C. for 12 hours.

1-5. Method of manufacturing spiral PCL structure with fine pattern

The bladder tissue has a helical muscle fiber structure. Thus, in order to fabricate a spiral PCL structure that mimics the bladder tissue, a PCL/PVA solution (PCL:PVA = 7:3, w/w) was used with a melt printing system having a 22G nozzle and a rotator of 500 rpm. A spiral PCL/PVA structure was manufactured by extrusion, and at this time, the moving speed of the nozzle was adjusted to 10 mm/s and the air pressure was adjusted to 250 kPa. Subsequently, the same lyophilization and washing processes as in the method of Example 1-4 were performed to prepare a spiral PCL structure.

1-6. Method for characterizing collagen and PCL structures

An image was acquired using SEM (SNE-3000M, SEC, Inc., Korea) to confirm the surface shape of the strut on which the fine pattern was formed. Sample preparation for image acquisition according to the manufacturer's instructions, before image acquisition, the fine patterned collagen structure was completely freeze-dried at -76 °C for 12 hours. In addition, the distribution of the orientation collagen fibers was measured using ImageJ software (National Institutes of Health, Bethesda, MD, USA). To compare the chemical structures of pure collagen, PVA, collagen / PVA mixture, and collagen from which PVA was removed, an FT-IR spectrometer (model 6700, Nicolet, West Point, PA, USA) was used, in which case, the IR spectrum was 500. Shows the average of 30 scans at -4000 cm ^-1 .

1-7. Rheological Measurement Method

Using a rotational rheometer (Bohlin Gemini HR Nano, Malvern Instruments, Surrey, UK) set to a conical-plate shape (diameter 40 mm, 4° cone angle, and 150 μm intervals), the complex viscosity ( n ^* ) and storage modulus (G') were measured. The 0.1-10 Hz dynamic frequency sweep range was performed with 1% strain at 25° C. in a linear viscoelastic region.

1-8. Cell culture

The patterned collagen network structure (7.5 × 3 × 0.5 mm ³ ) was sterilized by adding ultraviolet rays and 70% ethanol for 1 hour. C2C12 cells, myoblasts, are DMEM (Sigma-Aldrich, St. Louis) Manassas, containing FBS (Gemini Bio-Products, Sacramento, CA, USA) and 1% antibiotics (Antimycotic; Cellgro, Manassas, VA). VA) cultured in a medium under conditions of 37° C. and 5% CO ² . The medium was changed every 2 days, and DMEM medium containing 2% horse serum and 1% antibiotic was used to induce differentiation of myoblasts.

1-9. How to acquire fluorescence images

In each collagen construct, myoblasts were cultured for 1 and 3 days, and then stained with 0.15 mM calcein AM and 2 mM ethidium homodimer-1 at 37° C. for 30 minutes, and then live cells (green) and dead cells ( Red) was measured with a fluorescence microscope.

In addition, in order to acquire the myosin heavy chain (MHC) image of cells cultured in each collagen structure, the structure was washed twice with PBS after 7 and 14 days of culture, and the cells were 3.7% paraformaldehyde. It was fixed at room temperature by treatment with (paraformaldehyde). After fixing the cells, the construct was washed twice with PBS and immersed in 0.3% Triton X-100 for 30 minutes. Subsequently, after treatment with 1% BSA for 90 minutes, the sample was treated with rabbit anti-MHC antibody (Santa Cruz) diluted 1:50 and stored at 4° C. overnight. Secondary antibody conjugated with Alexa Fluor 488 (1:50, Molecular Probes) was treated at room temperature for 1 hour, 5 μM diamidino-2-phenylindole (DAPI; Invitrogen, Carlsbad, CA) and phalloidin (15 U/mL) (Invitrogen, Carlsbad, CA) was conjugated to Alexa Fluor 568 and counter-stained. Images were acquired using a CKX41 fluorescence microscope (Olympus), and ImageJ software was used to calculate the cell orientation (cell orientation).

1-10. Real-Time Polymerase Chain Reaction

In order to quantitatively measure the expression levels of myogenic markers Myod1, Myof5, Myogin, and Myh2, RT-PCR (Real-time polymerase chain reaction) was performed after 7, 14 and 21 days of culture. Total RNA for use in RT-PCR was isolated using a TRI reagent (Sigma-Aldrich), and the purity and concentration of the isolated RNA were measured with a spectrophotometer (FLX800T, Biotek, Winooski, VT, USA). CDNA was synthesized from RNase-free DNase-treated total RNA (500 ng) using a reverse transcription system, and the StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA) was used using Fast Universal PCR Master Mix (Applied Biosystems). qRT-PCR was performed. Myogenic marker expression was quantified using TaqMan Gene Expression Assays (Applied Biosystems). The marker information used is shown in Table 1 below.

Gene Catalog number beta actin (Actb) Mm00607939_s1 myogenic differentiation 1 (Myod1) Mm00435125_m1 myogenic factor 5 (Myf5) Mm00440387_m1 myogenin (Myog) Mm01332564_m1 myosin heavy chain 2 (Myh2) Mm00446194_m1

1-11. Statistical analysis

All data were expressed as mean ± standard deviation, statistical analysis was performed using SPSS (SPSS, Chicago, IL, USA), and analyzed by single factor variance (ANOVA). In all analyzes, * p <0.05, ** p <0.01 and *** p <0.001 were considered to indicate statistical significance.

Example 2. Analysis of optimal conditions for manufacturing collagen structures with fine patterns

2-1. Analysis of optimal PVA content for patterning fiber structure

In the preparation of the collagen structure, the effect of the PVA content on the micropattern and the optimal PVA content in the preparation of the cell culture support was investigated. To this end, the surface of the collagen structure prepared by the method of Example 1-3 was observed with a scanning electron microscope.

As shown in (a) to (d) in Figure 2, when the content of PVA is 15-20% by weight, the fiber bundle structure of the collagen structure was clearly shown after PVA was removed, and at this time, the diameter of the fiber structure was about It was confirmed that it was 1 to 2.7 μm. On the other hand, when using a low amount of PVA, it was confirmed that the pattern of the collagen structure appeared in a block format, which was not suitable for cell arrangement.

In addition, as a result of performing rheological analysis of the collagen structure prepared by the method of Example 1-3 by the method of Example 1-7, as shown in (e) to (g) in Figure 2, 20% by weight In the case of PVA exceeding, the viscosity was too high to mix with collagen, and 20% by weight of PVA from full width at half-maximum (FWHM) and complex viscosity (0.1-10 Hz) flowed from the nozzle during extrusion. It was found that the orientation is most appropriate for orientation. From the above, a polymer structure prepared using 20% by weight of PVA was analyzed below in order to form an appropriate microfiber of PVA.

2-2. Checking the fiber structure pattern of the collagen structure after removing PVA

To obtain collagen struts with a uniaxially arranged surface structure, a PVA fibrosis process was used, and the fibrous PVA was leached from the collagen/PVA mixture. FIG. 3A shows the surfaces of pure collagen struts (4 wt%), PVA/collagen struts before PVA leaching, and collagen struts from which PVA has been removed through a scanning electron microscope. As can be seen from the image of the surface and the comparison graph of the alignment distribution of FIG. 3B, the collagen strut from which PVA was removed showed a significantly higher degree of uniaxial alignment (FWHM: 14.7°) compared to the PVA/collagen strut and the pure collagen strut.

2-3. Analysis of optimal collagen/PVA mixing ratio for patterning fiber structure

Collagen and PVA were added to 7:3, 5:5, and 3:7, respectively, to determine the effect of the mixing ratio of collagen and PVA on the uniaxially arranged surface structure of the collagen strut and the optimal mixing ratio to obtain the uniaxially arranged surface structure. , And a collagen/PVA mixture mixed at 1:9 was used to prepare a collagen strut, and the degree of uniaxial alignment with the surface was confirmed. The collagen strut production method was the same as in Example 1-3 except for the mixing ratio of collagen and PVA.

As shown in Figure 4a, the strut produced by mixing collagen/PVA at a ratio of 7:3 did not show a uniaxial phase pattern, and in the case of other mixing ratios, a phase pattern in the shape of a uniaxially arranged fiber bundle was observed. Became. The above result is presumed that when the concentration of PVA is low, the process of forming fibrils of PVA is not smoothly performed. On the other hand, it was confirmed that the struts produced by mixing collagen/PVA at a ratio of 1:9 formed an unstable strut structure after PVA leaching, and the phases arranged uniaxially than the struts of the 5:5 mixing ratio and 3:7 mixing ratio It was confirmed that the pattern of was more clearly indicated, and it was determined that the mixing ratio of 5:5 was most appropriate for the manufacture of struts having a phase pattern of a uniaxially arranged fiber bundle structure. The following analysis was performed as a structure prepared by mixing collagen and PVA 1:1. .

2-4. Analysis of optimal flow rate when extruding mixtures for patterning fiber structures

Since the microfiber formation process of PVA is affected by wall shear stress, 0.02-0.11 μL is used to confirm the optimum flow rate (air pressure) when extruding the PVA/polymer mixture to obtain a uniaxially arranged surface structure. Collagen struts were prepared by varying the flow rate at /s and the degree of uniaxial alignment with the surface was confirmed.

As shown in FIG. 4B, it was found that the diameter of the strut increased as the pressure increased. In addition, collagen struts applied with low flow rates (0.02 and 0.05 μL/s) showed an unstable fiber pattern. This is presumed to be due to insufficient extrusion flow rate at the printer nozzle. On the other hand, it was confirmed that the increase in flow rate promotes alignment of the phase pattern on the collagen surface. Through analysis of the diameter of FWHM and struts according to various flow rates, it was found that a flow rate of 0.08 μL/s is most appropriate for the uniaxial arrangement of collagen struts.

Example 3. Analysis of Optimal Conditions for Manufacturing PCL Structures with Fine Patterns

In order to observe the effect of the mixing ratio of PCL and PVA on the uniaxial alignment structure, the air pressure applied to the printing nozzle was adjusted to 250 kPa, and a PCL structure was prepared using a PCL/PVC mixture mixed in various ratios, through a scanning electron microscope. The surface and cross-sectional images were obtained and shown in (d) to (f) in FIG. 5.

In Figure 5, (d) is a strut using pure PCL, (e) is a strut using a mixture of 7:3 PCL and PVA, and (f) is a mixture having the mixing ratio of 3:7. This is an image of the strut's surface and cross section. From the above, it can be seen that the strut using a mixture of PCL and PVA in a ratio of 7:3 shows the surface characteristics of the fiber bundle structure more, and as can be seen in (g) in FIG. 5 which analyzed the alignment level, the PCL structure It was found that the optimal PCL/PVA mixing ratio for manufacturing was 7:3.

Further, as shown in (h) to (j) in Fig. 5, it is possible to manufacture a PCL structure having a surface of a spiral fiber bundle structure such as the muscle tissue of the bladder by using the determined optimal PCL/PVA mixing ratio. Was confirmed.

Example 4. Culture of myoblasts on a polymeric cell scaffold with a fine pattern

4-1. Preparation of polymeric cell scaffolds with fine patterns

Collagen and PCL cell scaffolds having a uniaxially aligned fiber bundle structure for muscle tissue regeneration were prepared and their surface properties were confirmed. More specifically, a collagen structure with an aligned topological surface structure (CSAT) was prepared under the optimal conditions for formation of a fine pattern identified in Example 2 to perform an optical microscope and a scanning electron microscope. Was used to obtain an image of the surface. As a control, a conventional collagen structure (CCS) manufactured using a low-temperature printing process was used. The printing process was performed under the conditions of a working plate temperature: -10 °C, a moving speed of the printing nozzle: 20 mm/s, and a flow rate: 0.08 μL/s. The surface image of CCS is shown in Fig. 6(a), and the surface image of CSAT is shown in Fig. 6(b). As can be seen in (c) of Figure 6, the degree of uniaxial alignment (FWHM) was higher in CSAT.

4-2. Confirmation of cell proliferation and survival

In order to analyze the survival and proliferation of cells, C2C12 cells were cultured in CCS and CSAT, and after a certain period of time, MTT assay was performed, and images were obtained by fluorescent staining by the method of Example 1-9. Live cells are stained green and dead cells are stained red.

As a result, as shown in Figure 7a, both cells cultured in CCS and CSAT showed a high level of survival during the culture period. However, there was a difference in the morphology of the cultured cells. It was confirmed that the cells cultured on the CSAT cell support proliferated in the uniaxial direction coincident with the direction of the strut, as opposed to the cepons cultured on the CCS cell support and proliferating randomly. Could

4-3. Observation of cell nuclei and cytoskeleton

In order to observe the nuclei and cytoskeleton of C2C12 cells cultured in CCS and CSAT, the nucleus and F-actic were fluorescently stained by the method of Example 1-9 to obtain images. At this time, the nucleus is stained blue and the cytoskeleton is stained red.

As a result, as shown in Fig. 7b, the cytoskeleton of the cultured cells exhibited a multipolar structure, and in particular, in the case of the cells cultured on the CSAT cell support, it was confirmed that the cells were proliferated in the bipolar elongation of a structure aligned in parallel. .

Further, as a result of analyzing the alignment level (FWHM) of F-actin in the cultured cells using imageJ, as can be seen in (f) of FIG. 7b, CSAT is FWHM: 5.1° than CCS having FWHM of 28.5° The level of alignment was significantly higher.

4-4. Cellular MHC expression analysis and muscle cell marker gene expression analysis

It is known that MHC (Myosin heavy chain) is a protein of muscle fibers and is essential for the development of muscle cells. Accordingly, MHCs of C2C12 cells cultured in CCS and CSAT were fluorescently stained by the method of Example 1-9 to obtain an image and shown in FIG. 8A, and the alignment level of MHC was analyzed and shown in FIG. 8B.

As shown in FIGS. 8A and 8B, in the case of cells cultured on the CSAT cell scaffold after 7, 14, and 21 days of culture, the expression of MHC was higher than that of cells cultured in CCS, and the level of MHC alignment was also high. More specifically, after 14 days of culture, the MHC alignment level (FWHM) of the cells cultured in CSAT was 1.3°, the CCS was 20.9°, and the MHC alignment level (FWHM) of the cells cultured in CSAT 21 days after culture was 2.2°. , CCS was 31.1°. Particularly in the degree of cell proliferation at the 21st day after culture, CSAT proliferated to a level where the cells completely covered the cell support, but CCS did not.

In addition to the MHC alignment level analysis, the MHC positive region, the length and diameter of myotubes, the fusion index, and the maturity index were quantified and analyzed for comparison.

As shown in Fig. 8c, the MHC-positive region in the CSAT cell scaffold was significantly wider (f), and the diameter of the myotube did not show a significant difference from the CCS (h), but the length of the CSAT was longer (g). , In addition, in terms of the fusion index using the ratio of the number of nuclei in the MHC-positive region, the fusion index of CSAT was significantly higher than that of CSS (i), and from this, the CSAT structure was more effective in forming the root canal. It can be seen that it provides an effective platform. In addition, in the maturity index (j) comparing the ratio of myotubes with a total nucleus of 5 or more, CSAT showed a maturity of about twice or more compared to CCS.

In addition to the MHC analysis, the expression of Myod1, Myog, Myf5 and Myh2, which are genetic markers for evaluating differentiation into muscle cells (myogenesis) and differentiation into muscle fibers, was performed by RT-PCR in the manner of 1-10 in the above implementation. Quantified.

As shown in (k) to (m) of Fig. 8c, cells cultured in CSAT had a significantly higher expression level of the marker than in the case of CCS. These results suggest that the topological structure of CSAT provides a more rational platform to efficiently perform cell interaction and muscle development than that of CCS. That is, the above result means that the differentiation of myoblasts cultured in CSAT into muscle cells is promoted by a phase signal of a uniaxially arranged fiber bundle structure.

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 it 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 in all respects and not limiting.

Claims (11)

(a) preparing a PVA/polymer mixture by adding a biocompatible polymer to an amorphous polyvinyl alcohol (PVA) solution;
(b) extruding the PVA/polymer mixture to produce a single uniaxially arranged strut;
(c) lyophilizing the arranged PVA/polymer struts; And
(d) washing the PVA/polymer strut to remove PVA by leaching. A method for producing a cell scaffold having a fiber bundle structure.
The method of claim 1,
In the step (a), the PVA/polymer mixture is prepared by mixing a polymer and PVA in a ratio of 1 to 5: 5 to 9, wherein the method for producing a cell scaffold having a fiber bundle structure.
The method of claim 1,
The method for producing a cell scaffold having a fiber bundle structure, characterized in that the flow rate of the PVA/polymer mixture is 0.02 to 0.08 μL/s when extruded in step (b)
The method of claim 1,
The extrusion of the step (b) is a method of producing a cell support having a fiber bundle structure, characterized in that rotational extrusion.
The method of claim 1,
The manufacturing method further comprises the step of crosslinking the polymer by adding an EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) solution to the lyophilized PVA/polymer strut after step (c). A method for producing a cell support having a fiber bundle structure, characterized in that.
The method of claim 1,
The biocompatible polymer is a synthetic polymer or a natural polymer, characterized in that, the method for producing a cell support having a fiber bundle structure.
The method of claim 6,
The synthetic polymer is a group consisting of poly-ε-caprolactone (PCL), poly-lactic-co-glycolic acid (PLGA), and polylactic acid (PLLA). Method for producing a cell support having a fiber bundle structure, characterized in that at least one polymer selected from.
The method of claim 6,
The natural polymer is a method for producing a cell scaffold having a fiber bundle structure, characterized in that at least one polymer selected from the group consisting of collagen, chitosan, algnate, and gelatin.
A cell scaffold having a fiber bundle structure prepared by the method of claim 1.
A method for promoting differentiation of myoblasts into muscle cells, comprising culturing myoblasts on the cell support of claim 9.
The method of claim 10,
The method for promoting differentiation of myoblasts into muscle cells, characterized in that the muscle cells are skeletal muscle cells.

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