KR20220048511A - 3D artificial tubular-scaffold and manufacturing method thereof - Google Patents

Abstract

The present invention relates to a three-dimensional (3D) artificial tubular scaffold. The present invention provides a bio-implantable 3D artificial tubular scaffold and a manufacturing method therefor. More specifically, the present invention relates to a 3D artificial tubular scaffold having mechanical strength and flexibility suitable to be implanted in vivo, and a manufacturing method therefor. Furthermore, the invention provided herein relates to a 3D artificial tubular scaffold that performs a biological function besides a physical function in vivo, and a manufacturing method therefor.

Description

Three-dimensional artificial tubular scaffold and manufacturing method thereof {3D artificial tubular-scaffold and manufacturing method thereof}

The present disclosure relates to a three-dimensional artificial tubular scaffold that is implantable in vivo. Specifically, it relates to a three-dimensional artificial tubular scaffold having mechanical strength and flexibility suitable for in vivo implantation. Additionally, the disclosure herein relates to a three-dimensional artificial tubular scaffold that performs biological functions in addition to physical functions in vivo.

In the field of artificial scaffolds, research is being conducted to manufacture an artificial scaffold and implant it in a living body, and to replace a defective organ or organ function in the living body with the implanted scaffold.

At this time, research is being conducted on a method for manufacturing a scaffold having mechanical strength and flexibility suitable for implantation so that the artificial scaffold can be properly implanted in a living body.

In addition, research is being conducted to enable the artificial scaffold to perform a biological function in addition to a physical function replacing the function of a defective organ or organ. Specifically, research is being conducted to seed an artificial scaffold with cells capable of exhibiting the biological properties of an organ so that the cells can perform a biological function while differentiated and proliferated within the artificial scaffold.

The content disclosed in the present specification relates to an artificial scaffold having mechanical strength and flexibility suitable for in vivo implantation, an artificial scaffold suitable for cell adhesion, proliferation and differentiation, and a manufacturing method thereof, specifically in the field of artificial scaffold research as described above. it's about

An object of the present specification is to provide a three-dimensional artificial tubular scaffold having mechanical strength and flexibility suitable for in vivo implantation, and additionally performing biological functions in addition to physical functions in vivo.

The present specification provides a three-dimensional artificial tubular scaffold comprising the following. The artificial tubular scaffold includes an inner fiber layer made of a biodegradable polymer material in the form of fibers; A porous foam layer made of a biocompatible polymer material; and a fusion layer in which the biocompatible polymer material is inserted between the fibers of the fibrous layer and the two layers are fused, wherein the fibrous layer includes one or more cells and two or more fibers having different diameters, the porous foam layer includes It comprises one or more pores, and the fusion layer is characterized in that it is positioned between the inner fiber layer and the outer porous foam layer.

In one embodiment, the fibrous layer provides a three-dimensional artificial tubular scaffold comprising a fiber having a diameter of less than 1 micrometer and a fiber having a diameter of 1 micrometer or more.

In one embodiment, the cell is a three-dimensional artificial tubular scan, characterized in that it comprises hepatic progenitor cells reprogrammed by a medium composition for reprogramming human adult hepatocytes into hepatic progenitor cells comprising HGF, A83-01 and CHIR99021 provides folds.

In one embodiment, the three-dimensional artificial tubular scaffold provides a three-dimensional artificial tubular scaffold, characterized in that the artificial biliary tract.

In addition, the present specification provides a three-dimensional artificial tubular scaffold manufacturing method comprising the following steps.

The three-dimensional artificial tubular scaffold manufacturing method includes the steps of: (a) preparing a tubular 3D template using a water-soluble polymer; (b) electrospinning a biodegradable polymer material on a tubular 3D template in the form of fibers; (c) immersing the material made in step (b) in a biocompatible polymer solution in which the salt is dispersed; (d) immersing the material made in step (c) in water after drying; and (e) seeding the cells in the fibrous layer, wherein step (d) comprises salt leaching through water immersion and removal of the template prepared in step (a).

In one embodiment, the three-dimensional artificial tubular scaffold manufacturing method is characterized in that the water-soluble polymer material in step (a) is polyvinyl alcohol.

In one embodiment, the three-dimensional artificial tubular scaffold manufacturing method is characterized in that the polymer material of step (b) is PCL.

In one embodiment, the three-dimensional artificial tubular scaffold manufacturing method is characterized in that the polymer solution in step (c) is a polyurethane solution.

In one embodiment, the three-dimensional artificial tubular scaffold manufacturing method includes hepatic progenitor cells in which the cells are reprogrammed by a medium composition for reprogramming human adult hepatocytes into hepatic progenitor cells comprising HGF, A83-01 and CHIR99021 characterized in that

The three-dimensional artificial tubular scaffold provided herein has characteristics having mechanical strength and flexibility suitable for implantation in a living body. Additionally, there is an effect of performing a biological function in addition to the physical function in the living body.

1-3 is a diagram illustrating the process of forming an artificial biliary tract.
Figure 1a is the result of 3D design of the target biliary tract based on the MRI data, Figure 1b is the result of 3D printing according to the 3D model data to prepare a water-soluble template, Figure 2a is the process of forming a fibrous layer by electrospinning on the 3D template Figure 2b shows the process of forming a porous foam layer through salt leaching after dip coating by immersing a template in which a fiber layer is laminated in a salt-dispersed polymer solution.
Figures 3a-a show the overall shape of the final artificial scaffold, Figures 3a-b are results showing the cross-section of the scaffold consisting of a fibrous layer, a fusion layer, and a porous foam layer, Figures 3a-c are SEM images of the fibrous layer, Fig. 3a-d show SEM images of the porous foam layer. Scale bar is A, 20 mm; B, 50 μm, CD, 100 μm. Figure 3b shows the results of the restoring force test results by wrinkling and stretching (stretching), Figure 3c shows the tensile test results of the scaffold composed of a fiber layer, a fiber layer, and a foam layer.
4-5 are results of confirming the expression of biliary marker genes according to the differentiation of hCdHs.
Figure 4a shows the experimental design, Figure 4b shows the phase contrast (top) and double immunofluorescence staining images for the biliary markers CK19/CTFR and CK7/AE2 on the 14th day after differentiation induction compared to undifferentiated hCdH used as a negative control. , and the scale bar indicates 100 μm in black and 50 μm in white. Figure 4c shows the results of RT-qPCR analysis of the biliary tract marker gene at 14 days after differentiation, and Figure 5a is a fluorescence image showing the secretion of fluorescein diacetate indicative of bile acid delivery. Scale bar represents 100 μm. Figure 5b shows the results of heatmap and unsupervised hierarchical cluster analysis of whole gene expression profiles in human gallbladder tissue (black), hCdHs (blue) and hCdH-Chols (red).
6-7 are results comparing the differentiation of biliary tract cells of hCdH seeded and cultured on a flat dish and a fibrous scaffold.
Figure 6a shows the experimental design, Figure 6b shows representative phase contrast and live/dead staining images using calcein-AM (live, green) and propidium iodine (PI) (dead, red). hCdH was seeded on flat dishes and fibrous scaffolds and cultured in biliary tract cell differentiation medium for 14 days. Scale bar represents 500 μm. 7 shows the results of RT-qPCR analysis of proliferation, progenitor and biliary cell markers. Undifferentiated hCdH was used as a negative control and GAPDH was used as an internal control.
8-9 are results showing the difference in functional properties of hCdH-Chols according to the fiber structure.
Figure 8a shows the SEM image of the mat composed only of nanofibers and the nano, microfiber hybrid mat, and the scale bar represents 10 μm. Fig. 8b shows the fiber diameter distribution, and Fig. 8c shows a schematic diagram showing the more intimate cell-to-cell and cell-to-matrix interactions within the nano, microfiber hybrid mat (rough mat). Figure 9a shows SEM images of hCdHs and hCdH-Chols seeded on fine or coarse mats in reprogramming and biliary cell differentiation medium for 14 days, scale bar indicates 10 μm. Fig. 9b shows the results of RT-qPCR analysis of the biliary marker genes AE2, CFTR, AQP1 and CK19 in hCdHs and hCdH-Chols cultured on fine or coarse mats in reprogramming and biliary cell differentiation medium for 14 days, and Fig. 9c shows Double immunofluorescence staining images for the biliary marker proteins CK19/CFTR and CK7/AE2 are shown, and the scale bar indicates 50 μm.
10-11 are results showing that hCdH adheres, proliferates, and differentiates into biliary tract cells in the artificial biliary tract.
10a shows an immunofluorescence image stained with calcein-AM, and the scale bar indicates 500 μm. Figure 10b shows the double immunofluorescence staining results for the biliary tract markers CK19 (green) and CFTR (red) on day 14, and the scale bar shows 100 μm. 11 is a SEM image showing that densely packed cells are present only in the inner fibrous layer and not in the outer layer. Scale bar represents 200 μm.
12-13 show that mCherry immunofluorescence, postoperative status of rabbits, and hCdH-Chols populating 3D artificial biliary tract maintain expression of biliary tract cell lineage-specific marker proteins in vivo.
12A shows bright and mCherry fluorescence images of hCdH before seeding into artificial biliary tract. The scale bar represents 500 μm. 12B is a photograph of rabbits on days 1, 7, and 14 after surgery. 13a shows the results of H&E staining of the anastomotic site, and the scale bar indicates 500 μm. The boxed area represents the junction of the rabbit biliary tract and the 3D scaffold. 13B shows images of double immunotype and staining of mCherry (red) and the bile duct cell marker CK7 (green). Nuclei were counterstained with Hoechst. Scale bar represents 50 μm.
14-15 show the results of in vivo transplantation of an artificial biliary tract.
Fig. 14a shows the experimental design, and Fig. 14b shows photographs of the anastomosis site between the graft and the native common biliary tract immediately after transplantation (left) and 2 weeks after surgery (right). 15A shows an MRI image of a rabbit hepatobiliary tree at day 14, and arrows indicate anastomotic sites. Fig. 15b shows the results of double immunofluorescence staining of sections from the anastomosis site through the duodenum and hepatic side of the bile duct using CK19 (green) and mCherry (red) antibodies. The white dotted line delimits the boundary between the natural bile duct (NB) and artificial bile duct (AB). Scale bar represents 250 μm.

Problems of the prior art

In the conventional artificial scaffold for in vivo implantation, many studies have been conducted to prepare a scaffold using a biocompatible polymer material. Conventional artificial scaffolds were prepared by preparing a biocompatible polymer solution and then electrospinning to form a layer in the form of fibers. However, there was a problem in that the fiber form is easily broken and does not have sufficient mechanical strength necessary for the transplant operation procedure.

In addition, conventional artificial scaffolds have been manufactured to have a structure of various layers in order to improve the problems of physical properties. However, conventional scaffolds do not disclose the bonding between the layers. Accordingly, the conventional scaffold has a problem in that the scaffold layer may be separated. That is, the conventional scaffold has a problem in that delamination between the layers may occur due to the absence of a bonding relationship between the scaffold layers.

Finally, in the conventional artificial scaffold including cells, many studies have been conducted to attach the cells to the artificial scaffold. However, conventional scaffolds have been focused on finding a “specific” fiber diameter or pore size optimized for cell adhesion. However, a specific fiber diameter or pore size only provides the same size of space in the scaffold. The same space of these scaffolds makes it difficult for cells to freely penetrate and attach to the space within the scaffold. That is, the conventional scaffold has a problem in that cells are not attached to the space in the scaffold structure through various spaces, but are concentratedly attached to the surface of the scaffold. This problem has a limitation in not having an appropriate interaction between the cell and the cell and the cell and the matrix.

The present specification discloses a three-dimensional artificial tubular scaffold for solving the above problems and a method for manufacturing the same.

Construction of three-dimensional artificial tubular scaffolds

In general, in the case of a tubular scaffold for in vivo implantation, it must have adequate flexibility to withstand the pressure caused by the flow of the solution and to prevent damage to the scaffold due to movement after in vivo implantation. In addition, it must have adequate mechanical strength to prevent tearing due to sutures during transplantation surgery.

Additionally, in the case of a scaffold to which cells are attached, an appropriate environment must be provided so that the cells attached to the scaffold can perform biological functions while proliferating and differentiating in vivo.

In one aspect, the present specification provides a three-dimensional artificial tubular scaffold having the above appropriate flexibility, mechanical strength, and an appropriate environment in which cells can perform biological functions. The three-dimensional artificial tubular scaffold includes a fibrous layer, a fusion layer, and a porous foam layer.

The three-dimensional artificial tubular scaffold disclosed herein includes a fibrous layer.

The fiber layer is located at the innermost side of the three-dimensional artificial tubular scaffold, and includes fibers made of a biodegradable polymer material. The fiber layer is composed of fibers having various diameters. In one embodiment, the fibrous layer includes both a fiber having a diameter of a nanoscale and a fiber having a diameter of a microscale. In a preferred embodiment, the fibrous layer includes fibers having a diameter of less than 1 μm and fibers having a diameter of 1 μm or more. This fibrous configuration provides more sized spaces within the fibrous layer. Conventional scaffolds have been used in a form in which cells are intensively attached to the surface of the scaffold by providing a space of a consistent size. However, since the fibrous layer in the three-dimensional artificial tubular scaffold disclosed in the present specification has spaces of various sizes, cells penetrate deeper into the fibrous layer and are attached thereto.

According to Figure 8-9, it can be confirmed that cells survive on the surface in the fibrous layer consisting of only nanoscale fibers, but in the fibrous layer in which both nanoscale fibers and microscale fibers exist, cells form a cell population in the scaffold and survive. there is. In addition, due to these characteristics, the interaction between the cells and the cells and the cells and the matrix is improved, and it may have the effect of improving the differentiation efficiency of the cells.

The three-dimensional artificial tubular scaffold disclosed herein includes a porous foam layer.

The porous foam layer is located on the outermost part of the three-dimensional artificial tubular scaffold, and is made of a biocompatible polymer material. The porous foam layer includes one or more pores. The three-dimensional artificial tubular scaffold disclosed herein has a characteristic of flexibility due to the pores present in the porous foam layer. The pores may affect the mechanical strength and flexibility of the scaffold according to their size. At this time, the size of the pores may be adjusted to have mechanical strength and flexibility suitable for in vivo implantation.

In one embodiment, the pores are about 1-100 μm. In one embodiment, the size of the pores is about 10-90 μm. In one embodiment, the size of the pores is about 20 to 80 μm. In one embodiment, the size of the pores is about 30 to 70 μm. In one embodiment, the size of the pores is about 40-60 μm. In a preferred embodiment, the size of the pores is about 50 μm.

The three-dimensional artificial tubular scaffold disclosed herein has a feature that a porous foam layer is present in addition to the fibrous layer. In general, because the fibers are brittle and brittle, a scaffold comprising only a fibrous layer does not have adequate mechanical strength for an artificial tubular scaffold for implantation. However, the three-dimensional artificial tubular scaffold of the present specification has high mechanical strength by including a fibrous layer and a porous foam layer together. The porous foam layer functions to increase the mechanical strength of the scaffold by structurally supporting the broken and brittle fiber layer. Accordingly, the three-dimensional artificial tubular scaffold disclosed herein has mechanical strength suitable for in vivo implantation. According to FIG. 3B , the three-dimensional artificial tubular scaffold disclosed herein has mechanical strength capable of withstanding the physical manipulation of the in vivo implantation procedure.

In addition, the porous foam layer has a characteristic that the degree of flexibility is increased compared to the scaffold having only a fibrous layer. According to FIG. 3c, it can be seen that the fibrous layer and the porous foam layer exhibit greater flexibility with a lower elastic modulus and long elongation at break than those composed of only the fibrous layer.

The three-dimensional artificial tubular scaffold disclosed herein includes a fusion layer.

The fusion layer is located between the fiber layer and the porous foam layer, and the biocompatible polymer material constituting the porous foam layer is inserted between the fibers of the fiber layer.

The fusion layer has a function of firmly bonding the fiber layer and the porous foam layer to each other. Accordingly, the three-dimensional artificial tubular scaffold disclosed in the present specification has the effect of increasing mechanical strength and flexibility as the fibrous layer and the porous foam layer exist together, while also preventing the occurrence of peeling between the two layers. Simply stacking each layer for scaffold manufacturing may cause delamination in which each layer is separated because the bond between the layers is not strong. However, the three-dimensional artificial tubular scaffold disclosed herein has a strong bond between the layers due to the presence of a fusion layer in which a fiber layer and a porous foam layer are fused, so that it can function as a single scaffold without separation of each layer.

Three-dimensional artificial tubular scaffold material

In the three-dimensional artificial tubular scaffold disclosed herein, the fiber layer is made of a biodegradable polymer material.

In the three-dimensional artificial tubular scaffold disclosed herein, the fibrous layer in which cells are seeded should be capable of self-degrading after the scaffold is implanted in vivo. In one embodiment, after the scaffold is implanted in the living body, the fibrous layer is made of a biodegradable polymer material so that the fibrous layer self-decomposes and disappears after the cells grow and the tissue is restored. The biodegradable polymer material may be at least one selected from the group consisting of polycaprolactone, polyL-lactic acid, and polycarprolaction-co-lactide. However, the present invention is not limited thereto. One embodiment is polycarprolactone (PCL).

In the three-dimensional artificial tubular scaffold disclosed herein, the porous foam layer is made of a biocompatible polymer material.

The porous foam layer of the scaffold disclosed herein should not be decomposed or destroyed by itself in vivo because it must continuously perform a physical function after being implanted in vivo. In one embodiment, the porous foam layer of the scaffold disclosed herein is made of a non-degradable polymer. In addition, since the scaffold disclosed herein is for in vivo implantation, the porous foam layer is made of a biocompatible polymer material. The biocompatible, non-degradable polymer material is selected from the group consisting of polyethylene, polypropylene, polytetrafluoroethylene, poly(ethylene terephthalate), poly(methyl methacrylate), poly(2-hydroxyethyl methacrylate), poly(N-isopropylacrylamide), polysiloxane, polyurethanes It may be any one or more. However, the present invention is not limited thereto. One embodiment is polyurethanes.

The three-dimensional artificial tubular scaffold disclosed herein includes one or more cells in a fibrous layer.

The scaffold disclosed herein includes one or more cells in a fibrous layer to be implanted in vivo to perform a biological function as well as a physical function.

The cell may be a cell heterologous to the transplant target, an allogeneic cell, or an autologous cell. In one embodiment, the cell may be an autologous cell of a transplant target. When the three-dimensional artificial tubular scaffold of the present specification includes autologous cells of a transplant target, the scaffold can quickly adapt and function in vivo after being transplanted into a transplant target.

The cell may be a stem cell or a differentiated cell. When the three-dimensional artificial tubular scaffold disclosed herein includes stem cells, the stem cells differentiate into specific tissues according to cell signals around the implantation site in vivo so that the scaffold can perform biological functions in vivo. do. When the three-dimensional artificial tubular scaffold disclosed herein includes differentiated cells, the differentiated cells may be appropriately selected according to the implantation site of the scaffold. In one embodiment, the cells are embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells, placental stem cells, retrodifferentiated stem cells, endothelial cells, epithelial cells, skin cells, endoderm cells, mesoderm cells, fibroblasts, bone It may be any one or more selected from the group consisting of cells, chondrocytes, hepatocytes, liver progenitor cells, biliary tract cells, pancreatic cells, and the like. However, the present invention is not limited thereto. One embodiment is a biliary tract cell. One embodiment is a liver progenitor cell.

In one embodiment, the cell may be a reprogrammed cell after extraction from the transplant target. In one embodiment, the cell may be a cell extracted from a transplant target and differentiated after reprogramming. In one embodiment, the cells may be liver progenitor cells reprogrammed from adult human hepatocytes. In one embodiment, the cells may be biliary duct cells differentiated from hepatic progenitor cells reprogrammed from adult human hepatocytes. In one embodiment, the cell may be a hepatic progenitor cell reprogrammed by a medium composition for reprogramming human adult hepatocytes into hepatic progenitor cells comprising HGF, A83-01 and CHIR99021. In one embodiment, the cells may be biliary duct cells differentiated from hepatic progenitor cells reprogrammed by a medium composition for reprogramming human adult hepatocytes into hepatic progenitor cells comprising HGF, A83-01 and CHIR99021.

Functions of 3D Artificial Tubular Scaffolds

Carrying out physical functions in vivo

The three-dimensional artificial tubular scaffold disclosed herein can perform a physical function by replacing a defective organ in vivo. The three-dimensional artificial tubular scaffold disclosed herein can be implanted in a living body due to its appropriate mechanical strength and flexibility to function to maintain a normal flow of in vivo material. In one embodiment, the three-dimensional artificial tubular scaffold disclosed herein may function to have a normal flow of bile.

performing biological functions in vivo

The three-dimensional artificial tubular scaffold disclosed herein can perform biological functions after cells are attached to it and implanted in a living body. Cells attached to the artificial scaffold can perform a biological function while proliferating and differentiating in vivo, thereby helping the scaffold to function well in vivo. In one embodiment, the three-dimensional artificial tubular scaffold disclosed herein may perform a function of tissue regeneration in vivo by providing an environment for cell attachment, proliferation, and differentiation. In one embodiment, the three-dimensional artificial tubular scaffold disclosed herein may function to regenerate the biliary tract tissue.

Uses of three-dimensional artificial tubular scaffolds

The three-dimensional artificial tubular scaffold disclosed herein may be used for therapeutic purposes or experimental purposes. However, it is not limited to a therapeutic purpose or an experimental purpose, and may be used according to other suitable uses.

therapeutic use

The three-dimensional artificial tubular scaffold disclosed herein can be implanted in a subject in need of implantation. In one embodiment, the three-dimensional artificial tubular scaffold disclosed herein may be used in an animal in need of transplantation. In one embodiment, the three-dimensional artificial tubular scaffold disclosed herein may be used in a human in need of implantation.

In one embodiment, the three-dimensional artificial tubular scaffold may be provided as an organ or organ for in vivo implantation. Specifically, the three-dimensional artificial tubular scaffold disclosed herein may be used when an organ or organ in a living body is damaged or has a defect in its function and it is necessary to replace the organ or organ. In one embodiment, the three-dimensional artificial tubular scaffold disclosed herein may be used to replace a tubular organ or organ in vivo. In one embodiment, the three-dimensional artificial tubular scaffold disclosed herein may be used to replace a trachea, biliary tract, esophagus, blood vessel, and the like. In one embodiment, the three-dimensional artificial tubular scaffold disclosed herein may be used to replace the biliary tract in vivo.

experimental use

In one embodiment, the three-dimensional artificial tubular scaffold disclosed herein may be used for experimental purposes. In one embodiment, the three-dimensional artificial tubular scaffold disclosed herein may be implanted in a non-human animal, and may be used to evaluate the effect of implantation by comparing an implanted non-human animal with a non-transplanted non-human animal.

Features of three-dimensional artificial tubular scaffolds

First, the three-dimensional artificial tubular scaffold disclosed herein has a fibrous layer and a porous foam layer.

The porous foam layer of the three-dimensional artificial tubular scaffold disclosed herein can mechanically support the fragile fibrous layer while maintaining structural flexibility. In addition, the porous foam layer includes one or more pores, and the pores present in the porous foam layer allow the scaffold to have structural flexibility. That is, the three-dimensional artificial tubular scaffold disclosed herein has mechanical strength and flexibility suitable for in vivo implantation.

The three-dimensional artificial tubular scaffold disclosed herein has appropriate mechanical strength and flexibility due to the presence of the fibrous layer and the porous foam layer together, so that the thickness of the scaffold can be appropriately manufactured. In general, in order to increase the mechanical strength of the scaffold, a method of manufacturing a thick scaffold is used. The three-dimensional artificial tubular scaffold of the present specification has sufficient mechanical strength and flexibility while having an appropriate thickness. In one embodiment, the thickness of the three-dimensional artificial tubular scaffold of the present disclosure is less than about 600 micrometers. In one embodiment, the thickness is less than about 550 micrometers. In one embodiment, the thickness is less than about 500 micrometers. In a preferred embodiment, the thickness is greater than or equal to about 440 micrometers and less than 470 micrometers.

Second, the three-dimensional artificial tubular scaffold disclosed herein has a fusion layer.

The three-dimensional artificial tubular scaffold disclosed herein is characterized in that a fusion layer exists between the fibrous layer and the porous foam layer. The fusion layer exists in a form in which the biocompatible polymer material constituting the porous foam layer is inserted between the fibers of the fiber layer. The fusion layer serves to strengthen the bond between the fiber layer and the porous foam layer. The fusion layer serves to prevent the occurrence of separation between the fibrous layer and the porous foam layer so that the layer structure of the scaffold is not separated and functions as one scaffold.

Third, the three-dimensional artificial tubular scaffold disclosed herein includes fibers of various diameters in a fibrous layer.

The three-dimensional artificial tubular scaffold disclosed herein is characterized in that the diameter of the fibers constituting the fibrous layer is diversified. The fibrous layer containing fibers of different diameters serves to vary the size of the space within the fibrous layer. The space in the fibrous layer serves to allow cells to freely penetrate into and attach to the fibrous layer structure. The space within the fibrous layer serves to increase cell-to-cell and cell-to-scaffold interactions. In addition, the fibrous layer in which nano-scale fibers and micro-scale fibers coexist has a feature of greatly improving cell differentiation efficiency by increasing porosity.

Finally, the three-dimensional artificial tubular scaffold disclosed herein includes one or more cells in a fibrous layer to perform a biological function.

The three-dimensional artificial tubular scaffold disclosed herein includes one or more cells in a fibrous layer. The cells may be attached to the fibrous layer in the scaffold to proliferate and differentiate. The cell may perform a biological function after the scaffold is implanted in a living body. The cell may function so that the tissue can be regenerated in vivo after the scaffold is implanted in the living body.

Manufacturing method of three-dimensional artificial tubular scaffold

The present specification provides a method for manufacturing a three-dimensional artificial tubular scaffold. The manufacturing method disclosed herein may include the following steps.

The method is an example,

(a) preparing a tubular 3D template using a water-soluble polymer material;

(b) Electrospinning a biodegradable polymer material on a tubular 3D template in the form of fibers;

(c) (b) immersing the material made in step in a biocompatible polymer solution in which the salt is dispersed;

(d) (c) immersing the material made in step in water after drying; and

(e) Seeding the cells into the fibrous layer.

As a further example, the manufacturing method may additionally include a surface treatment step of the 3D template between steps (a) and (b).

As a further example, the manufacturing method may additionally include a preparation step before seeding the cells in the fibrous layer after step (d).

Hereinafter, each step will be described in detail.

Steps to prepare a tubular 3D template from a water-soluble polymer material

In order to prepare a scaffold structure suitable for implantation, a 3D template can be prepared from a water-soluble polymer material.

The 3D template may be manufactured using a mold suitable for a transplant organ or organ structure, or manufactured according to a 3D printing method. In an embodiment, the 3D template may be manufactured according to a 3D printing method.

A 3D template can be manufactured according to a 3D printing method using design data. The 3D template may be manufactured according to a 3D printing method using 2D design data or 3D design data. In an embodiment, the 3D template may be manufactured according to a 3D printing method using magnetic resonance image data (MRI). In an embodiment, the 3D template may be manufactured according to a 3D printing method using computer-aided design (CAD) based on magnetic resonance image data.

The 3D template may be manufactured by 3D printing according to a photopolymerization method, a material injection method, a binder injection method, a material extrusion method, a powder fusion method, a sheet lamination method, or a directional energy deposition method. In an embodiment, the 3D template may be manufactured by 3D printing according to a material extrusion method.

A water-soluble polymer material is used to manufacture the 3D template. Water-soluble polymer materials that can be used for 3D template manufacturing include PVA (polyvinyl alcohol), PVP (polyvinyl pyrrolidone), PEO (polyethylene oxide), CMC (carboxyl methyl cellulose), starch, PAA (polyacrylic acid) and hyaluronic acid ( Hyaluronic acid) may be any one or more selected from the group consisting of. However, the present invention is not limited thereto. In an embodiment, the water-soluble polymer material is polyvinyl alcohol.

For 3D template fabrication, the appropriate 3D printer nozzle inner diameter, nozzle temperature, and lamination thickness can be set. In an embodiment, the nozzle inner diameter, the nozzle temperature, and the lamination thickness are about 0.1 to 0.4 mm, 150 to 190° C., and 0.05 to 0.25 mm, respectively. In an embodiment, the nozzle inner diameter, the nozzle temperature, and the lamination thickness are about 0.2 to 0.3 mm, 160 to 180° C., and 0.1 to 0.2 mm, respectively. In one embodiment, the nozzle inner diameter, the nozzle temperature, and the lamination thickness are about 0.25 mm, 170° C., and 0.15 mm, respectively.

Surface treatment steps in 3D template

The step of smoothing the surface of the manufactured 3D template may be optionally performed. A physical method or a chemical method may be used to smooth the surface of the 3D template. In order to smooth the surface of the 3D template, mechanical machining, ultrasonic machining, laser machining, electric discharge machining, electrolytic machining, etc. may be used. In one embodiment, an ultrasonic machining method is used to smooth the surface of the 3D template.

For example, to smooth the surface of the 3D template, the template can be immersed in distilled water and then sonicated. At this time, distilled water is about 40 to 60 ℃ in a specific embodiment, distilled water is about 50 ℃. In addition, the time for immersing the template in distilled water is about 30 seconds to 2 minutes. In one embodiment, the time for immersing the template in distilled water is about 1 minute.

Depositing a biodegradable polymer material in the form of fibers on the surface of the 3D template

In order to deposit a biodegradable polymer material in the form of a “fiber” on the surface of the 3D template, the biodegradable polymer material must be prepared in the form of a fiber. Various spinning methods may be used to prepare the biodegradable polymer material in the form of fibers. In one embodiment, an electrospinning method may be used to prepare the biodegradable polymer material in the form of fibers.

In order to deposit a biodegradable polymer material on the surface of the 3D template, a biodegradable polymer solution must be electrospun on the “surface” of the 3D template. In one embodiment, the biodegradable polymer solution may be electrospinning on the 3D template surface while rotating the 3D template to deposit the biodegradable polymer material on the 3D template surface.

The biodegradable polymer solution may be prepared by dissolving the biodegradable polymer material in an appropriate solvent. The biodegradable polymer material is at least one material selected from the group consisting of polycaprolactone, polyL-lactic acid, and polycarprolaction-co-lactide. However, the present invention is not limited thereto. The suitable solvent may be one or more of the organic solvents.

In one embodiment, the biodegradable polymer solution may be prepared by dissolving polycarprolactone in a solvent mixture of methylene chloride and dimethylformaldehyde. In one embodiment, the biodegradable polymer solution was prepared by dissolving polycarprolactone in a solvent mixture of methylene chloride and dimethylformaldehyde in a ratio of 3:1.

A biodegradable polymer material can be deposited in the form of fibers having various diameters on the surface of the 3D template. The concentration of the biodegradable polymer solution can be adjusted to deposit the biodegradable polymer material in the form of fibers having various diameters on the 3D template surface. In one embodiment, a high concentration biodegradable polymer solution may be used. In one embodiment, the concentration of the high-concentration biodegradable polymer solution may be about 5 to 30% (w/v). In one embodiment, it may be 8 to 25% (w/v). In one embodiment, it may be 15 to 22% (w/v). In a preferred embodiment, it is 18% (w/v).

Step of immersing the fiber-deposited 3D template in a biocompatible polymer solution

To prepare a porous foam layer on the fiber-deposited 3D template, the fiber-deposited 3D template is immersed in a biocompatible polymer solution. In this process, a fusion layer in which the biocompatible polymer solution is inserted between the fibers of the fiber layer and fused may be formed. The fusion layer generated in this process can serve to firmly bond the fiber layer and the porous foam layer to be generated later.

Various methods of creating pores to prepare the porous foam layer can be used. In an embodiment, a salt leaching method, a gas foaming method, or the like may be used as a method for generating pores. In an embodiment, a salt leaching method may be used as a method of generating pores. In one embodiment, in order to prepare a porous foam layer, a 3D template on which fibers are deposited may be immersed in a biocompatible polymer solution in which salt is dispersed.

The biocompatible polymer solution in which the salt is dispersed is prepared by mixing salt particles in the biocompatible polymer solution.

The biocompatible polymer solution is prepared by dissolving a biocompatible polymer material in an appropriate solvent. The biocompatible polymer material is polyethylene, polypropylene, polytetrafluoroethylene, poly(ethylene terephthalate), poly(methyl methacrylate), poly(2-hydroxyethyl methacrylate), poly(N-isopropylacrylamide), polysiloxane, any one or more selected from the group consisting of polyurethanes can However, the present invention is not limited thereto. The suitable solvent may be one or more of the organic solvents. In one embodiment, the biocompatible polymer solution may be prepared by dissolving thermoplastic polyurethane in dimethylformaldehyde. In one embodiment, the concentration of the solution is about 5-25% (w/v). In one embodiment, the concentration of the solution is about 10-20% (w/v). In one embodiment, the concentration of the solution is about 15% (w/v).

The biocompatible polymer solution in which the salt is dispersed is prepared by mixing salt particles having a property of being leached by a solvent to the biocompatible polymer solution. In one embodiment, the solvent is water. In an embodiment, the salt may be any one or more of salts having a property of reacting with water and leaching. However, the present invention is not limited thereto. In one embodiment, the salt is NaCl. In one embodiment, a 400% (w/w) NaCl solution was used as the biocompatible polymer solution in which the salt was dispersed.

The pore size of the porous foam layer can be adjusted by changing the conditions of the biocompatible polymer solution in which the salt is dispersed. In one embodiment, the pore size of the porous foam layer may be adjusted by changing the salt particle size of the biocompatible polymer solution in which the salt is dispersed. In one embodiment, the salt particle size is greater than or equal to about 30 micrometers. In one embodiment, the salt particle size is greater than or equal to about 40 micrometers. In one embodiment, the salt particle size is greater than or equal to about 45 micrometers. In one embodiment, the salt particle size may be adjusted by sieving with a mesh. In one embodiment, the salt particle size may be about 40-50 micrometer mesh sieved salt particles. In one embodiment, the salt particles are salt particles sieved to about 45 micrometer mesh.

A step of immersing a template immersed in a salt-dispersed biocompatible polymer solution in water after drying

The template immersed in the biocompatible polymer solution in which the salt is dispersed is immersed in water after drying. In an embodiment, the template may be immersed in water so that salt particles may be leached out. In one embodiment, the template may be immersed in water for about 1 minute or more in order to leach out the salt particles. In a preferred embodiment, the template may be immersed in water for about 2 minutes to allow the salt particles to leach out. In an embodiment, the water may be ultrasonicated water. Through this process, a porous foam layer including pores may be formed on the template.

The template immersed in the salt-dispersed biocompatible polymer solution is immersed in water after drying, so that the first 3D template prepared using a water-soluble polymer material can be removed. In one embodiment, the dried template may be immersed in water for about 10 minutes or more after being immersed in the biocompatible polymer solution in which the salt is dispersed. In one embodiment, the template may be immersed in water for about 20 minutes or more. In a preferred embodiment, the template is immersed in water for about 30 minutes. In one embodiment, the template may be immersed in water at about 30 ~ 70 ℃. In one embodiment, the template may be immersed in water at about 40 to 60 °C. In a preferred embodiment, the template is immersed in water at about 50°C. In an embodiment, the water may be ultrasonicated water.

The process in which the template immersed in the biocompatible polymer solution in which the salt is dispersed is dried and then immersed in water to leach the salt. can In an embodiment, salt leaching and removal of the first 3D template manufactured using a water-soluble polymer material may occur simultaneously through the water immersion step. In an embodiment, the first 3D template manufactured using a water-soluble polymer material may be removed after salt leaching through a water immersion step.

After drying the template immersed in the biocompatible polymer solution in which the salt is dispersed, the method may further include cutting the tip of the template before or during the step of immersing in water. When the end of the template is cut and immersed in water, the first 3D template prepared using a water-soluble polymer material can be removed more effectively.

Preparatory steps before seeding cells on the fibrous layer

A preparatory step before seeding the cells may optionally be included prior to the step of seeding the cells into the fibrous layer. In one embodiment, it may include one or more of washing, sterilizing, or coating the scaffold before seeding the cells. In one embodiment, it may include washing the scaffold with ethanol before seeding the cells. In one embodiment, it may include sterilizing the scaffold with UV light before seeding the cells. In one embodiment, it may include washing the scaffold with ethanol before seeding the cells and then sterilizing with UV light. In one embodiment, it may include coating the scaffold with gelatin before seeding the cells. In one embodiment, it may include a step of washing with ethanol before seeding and then coating with gelatin. In one embodiment, it may include the step of coating the scaffold with gelatin after the step of sterilizing with ultraviolet light before seeding. In one embodiment, the scaffold may be coated with about 0.1% gelatin. In one embodiment, the scaffold may be coated with about 0.1% gelatin and incubated for 30 min at 37 °C and 5% CO2. The coating with gelatin may include removing excess gelatin after coating and culturing with gelatin and drying at room temperature.

Seeding the cells into the fibrous layer

The three-dimensional artificial tubular scaffold manufacturing method disclosed herein may include seeding cells after drying a template immersed in a salt-dispersed biocompatible polymer solution and immersing it in water. The cells may be seeded on a fibrous layer in an artificial tubular scaffold.

The cell may be a cell derived from the same species as the cell to be transplanted, or a cell derived from a different species. In one embodiment, the cell may be a cell derived from a transplant target.

The cells may be stem cells or differentiated cells. In one embodiment, the cells are embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells, placental stem cells, retrodifferentiated stem cells, endothelial cells, epithelial cells, skin cells, endoderm cells, mesoderm cells, fibroblasts, bone It may be any one or more selected from the group consisting of cells, chondrocytes, hepatocytes, liver progenitor cells, biliary tract cells, pancreatic cells, and the like. However, the present invention is not limited thereto. One embodiment is a liver progenitor cell. One embodiment is a biliary tract cell. In one embodiment, hepatic progenitor cells are reprogrammed from adult human hepatocytes. One embodiment is a biliary tract cell differentiated from hepatic progenitor cells reprogrammed from adult human hepatocytes. One embodiment is a liver progenitor cell reprogrammed by a medium composition for reprogramming human adult hepatocytes into hepatocytes comprising HGF, A83-01 and CHIR99021.

Uses of the three-dimensional artificial tubular scaffold manufacturing method

The three-dimensional artificial tubular scaffold manufacturing method disclosed herein may be used for manufacturing an organ or organ-specific scaffold to be implanted in vivo. However, the present invention is not limited thereto and may be used according to other suitable uses.

The three-dimensional artificial tubular scaffold disclosed herein may be used to manufacture a scaffold suitable for an organ or organ size and structure suitable for in vivo implantation. The three-dimensional artificial tubular scaffold manufacturing method disclosed in the present specification can be 3D designed using magnetic resonance imaging of an organ or organ to be transplanted, and a 3D template of a custom size and structure for transplantation can be manufactured through 3D printing using the same. Through the prepared 3D template, a three-dimensional artificial tubular scaffold having an implant-customized size structure can be manufactured.

The three-dimensional artificial tubular scaffold disclosed herein may include seeding cells suitable for a transplant target and an organ or organ to be transplanted. In an embodiment, the artificial tubular scaffold including cells derived from a transplantation target can quickly adapt after being transplanted into a transplantation target and perform its function in vivo. In an embodiment, an artificial tubular scaffold including cells tailored to an organ or organ to be transplanted may be transplanted in vivo to perform a biological function according to the transplanted organ or organ.

Example

Hereinafter, the present invention will be described in detail through examples.

At this time, the examples are only for explaining the present invention in more detail, and it is obvious to those of ordinary skill in the art that the scope of the present invention is not limited by these examples.

experimental method

MRI image

MRI was performed 1 day before and 14 days after surgery using a 3T MRI scanner with a small distal coil (Ingenia; Philips Healthcare, Best, The Netherlands). Rabbits were anesthetized with intramuscular injections of ketamine (40-100 mg/kg) and xylazine (5-13 mg/kg) during MRI scans. Briefly, three-sided localized imaging gradient echo sequences were first obtained, followed by respiratory induced 3D magnetic resonance cholangiopancreatography (MRCP) images and thick slab single-shot 2D MRCP images. The acquired 3D MRCP images were saved in Digital Imaging and Communication Medicine (DICOM) format and processed into 3D objects in a 3D printable file format (STL, Surface Tessellation Language) using commercial software (Mimics; Materialize, Louvain, Belgium). .

Biliary duct cell differentiation

This experiment was performed according to the protocol approved by the institutional review committee (HYI-16-229-3) of Hanyang University. Human liver tissues were collected from three donors who underwent surgery at Hanyang University Medical Center with the consent of the patient. The generation of hCdH is determined by Y. Kim, K. Kang, S. B. Lee, D. Seo, S. Yoon, S. J. Kim, K. Jang, Y. K. Jung, K. G. Lee, V. M. Factor, J. Jeong, D. Choi, J Hepatol 2019 , 70, 97. Briefly, primary human hepatocytes were isolated using collagenase and seeded on collagen-coated plates (STEMCELL Technologies, BC, Canada) in William's E medium (Gibco, CA, USA). The next day, the medium was reprogrammed with DMEM/F12 (Gibco, CA, USA) supplemented with 20 ng/ml hepatocyte growth factor (HGF) (Peprotech), 3 μM CHIR99021 (STEMCELL Technologies) and 4 μM A83-01 (Gibco) reprogramming medium (hereinafter HAC). ) was changed to hCdHs were produced within 1 week. To induce differentiation of hCdH into biliary tract cells (hCdH-Chols), the protocol described in E. M. Buisson, J. Jeong, H. J. Kim, D. Choi, Int J Stem Cells 2019, 12, 183. was used. Briefly, hCdH was treated with DMEM/F12 supplemented with 7.5 ng/ml epidermal growth factor (EGF), 7.5 μM CHIR and 7.5 ng/ml HGF for 2 weeks and 10 μM sodium taurocholate hydrate (Sigma) for the last 2 days. Biliary duct cells were seeded into gelatin (Sigma)-coated dishes in differentiation medium (CDM) containing

immunofluorescence staining

Cells, scaffolds and biliary tract were fixed in 4% paraformaldehyde for 20 min or overnight, respectively. Staining was performed according to the manufacturer's recommendations. Nuclei were stained with Hoechst 33342 (1:10000, Molecular Probes, USA). Cells were imaged using a TCS SP5 confocal microscope (Leica, Germany).

Antibody List

mRNA isolation and RT-qPCR analysis

Total RNA was isolated using TRIzol reagent (Ambion, Foster City, CA, USA) and chloroform. Reverse transcription of 1 μg of RNA was performed using the Transcriptor First Strand cDNA Synthesis Kit (Roche, Mannheim, Germany). To perform real-time polymerase chain reaction (RT-qPCR), oligonucleotide primers were used along with 1 μg of cDNA and 10 μg of PCR PreMix (Dyne bio, Seongnam, Korea). The PCR cycle was 20 seconds at 95 °C and 40 seconds at 60 °C, a total of 40 cycles.

Primer list

Fluorescein diacetate (FD) analysis

Cells were washed with PBS and incubated with fluorescein diacetate (Sigma) diluted in medium at 37 °C for 15 minutes at a concentration of 2.5 μg/mL. After the FD-containing medium was removed, the cells were incubated in fresh medium for an additional 30 minutes. Fluorescence images were taken immediately after incubation in FD-containing medium and after replacing fresh medium with Hanks Buffer (HBSS).

Live/Dead staining

The medium was removed and the cells were washed with PBS. To tag cells with Calcein (live staining) or Propidium Iodide (dead staining) (ThermoFisher), 1 μl of calcein and 1 μl of proprium iodide were added to 1 ml of medium, and cells, scaffolds and artificial biliary tract fragments were placed at 37 °C. and 5% CO2 for 30 min. The medium was then removed and replaced with fresh medium. Afterwards, pictures were taken using a fluorescence microscope.

cell seeding

Before cell seeding, the fibrous scaffold and artificial biliary tract were cut, washed with ethanol, and then sterilized with UV light. After 30 min, the fibrous scaffold and artificial biliary tract were coated with 0.1% gelatin (Sigma) and incubated at 37 °C and 5% CO2 for 30 min. The excess gelatin was then removed and the sample dried at room temperature for 2 hours. hCdH was passaged using previously established methods. 106 cells were suspended in 10 μl of HAC medium and seeded inside the artificial biliary tract construct. They were then incubated at 37 °C and 5% CO2 for 1 h. In a 4-well plate, 0.5 ml of reprogramming medium per well was used and replaced with CDM medium the next day.

SEM imaging

The microstructure of the electrospun fibers and cultured cells was visualized using a scanning electron microscope. The scaffolds with cells were washed 3 times with PBS and fixed in 5% glutaraldehyde solution containing 0.1M sodium cacodylate and 0.1M sucrose for 30 min. After washing with distilled water for 5 minutes, samples were slowly dehydrated by continuous incubation in 50%, 60%, 70%, 80%, 90%, and 100% ethanol solutions for 5 minutes each, followed by hexamethyldisilazane (HMDS; JT Baker, Phillipsburg, NJ, USA) for 15 min. Upon complete drying, the samples were sputtered with Pt until a 20 nm thick coating was obtained. Samples were imaged via SEM (S-4800; Hitachi, Japan).

Library preparation and transcriptome sequencing

Total RNA concentration was calculated by Quant-IT RiboGreen (Invitrogen, #R11490) and integrity values were accessed by TapeStation RNA screen tape (Agilent, #5067-5576). Only high-quality RNAs with RNA Integrity Number (RIN) greater than 7.0 were used for RNA library construction. Libraries were independently prepared using 1 ug of total RNA for each sample by Illumina TruSeq Stranded mRNA Sample Prep Kit (Illumina, Inc., San Diego, CA, USA, # RS-122-2101). An initial step in library preparation involved purifying poly-A containing mRNA molecules using poly-T attached magnetic beads. After this step, the purified mRNA was fragmented into small fragments using divalent cations at elevated temperature. The cleaved mRNA fragment was copied to the top strand of cDNA using SuperScript II reverse transcriptase (Invitrogen, # 18064014) and random primers, and the reverse complementary strand of cDNA was synthesized using DNA polymerase I, RNase H and dUTP. . These cDNA fragments underwent an end repair process by adding a single 'A' base and an adapter bond. After this step the final cDNA library was generated and amplified by PCR. The generated library was quantified using the KAPA Library Quantification kit for Illumina Sequencing platform according to the qPCR Quantification Protocol Guide (KAPA Biosystems, #KK4854), and verified by TapeStation D1000 ScreenTape (Agilent Technologies, #5067-5582). Indexed libraries were paired-end sequenced by an Illumina Hiseq2500 from Macrogen Inc. (Illumina, Inc., San Diego, CA, USA).

Cryosectioning and H&E staining

Samples were frozen at -20 °C in OCT and frozen sectioned into 4 μm thick slices. Slides were fixed in 95% alcohol for 5 min and stained with H&E.

Bioinformatics analysis

Illumina standard pipeline and real-time analysis (RTA) tools were used for raw image processing, base calling and It was used to generate FASTQ data. 101bpX2 read sequences were preprocessed using Sickle (v1.33) (32) to trim bad subsequences or CutAdapt (v1.5) (33) to remove adapter sequences. Sequencing reads after pretreatment were performed using RSEM (v1.2.31) with STAR (v2.5.2b) alignment software (34,35) to obtain the Hg19 human reference genome (University of California, University of California iGenomes, https://support. (available at illumina.com/sequencing/sequencing_software/igenome.html). By measuring the amount of the expression level of a transcript as a TPM (Transcripts Per Kilobase Million) score, the proportion of reads mapped to a gene in each sample can be easily compared. Raw sequencing data is provided from GEO (https://www.ncbi.nlm.nih.gov/geo) under the number GSE152809. Clustering analysis was performed using Cluster3.0 (Michael Eisen lab, http://eisenlab.org/) and visualization was performed with R software (v3.3.2, https: www.r-project.org).

in vivo transplantation

Animal experiments were performed in accordance with NIH guidelines and Hanyang University Animal Research Protocol with permission from Hanyang University IACUC (Institutional Animal Care and Use Committee No. 2018-0097A). Dooyeol Biotech (Seoul, Republic of Korea) provided 21-month-old female semi-specific-pathogen-free rabbits weighing 3-4 kg. All animals were housed in standard conditions with a 12-hour light-dark cycle and fed a standard diet for a 4-week acclimatization period. Rabbit was fasted for 12 hours before surgery. The animals were immobilized in a supine position under general anesthesia administered by an anesthesiologist and laparotomy was performed through a midline incision in the epigastrium to expose the common bile duct (CBD). The lower CBD was dissected and the artificial biliary tract was anastomosed to the proximal and distal ends of the CBD with 10-0 Ethilon® cut sutures under light microscopy. All procedures were performed by a microsurgeon. Adequate bile drainage through an anastomotic artificial biliary tract was confirmed during surgery. Before closing, the abdominal cavity was washed with warm saline. For routine histology, samples of rabbit liver and reconstituted biliary tract were immediately placed in 10% neutral buffered formalin and fixed at 4 °C for 24 h. Tissues were processed, embedded in paraffin, sectioned at 4 μm, and stained with hematoxylin and eosin (H & E). Histopathological examination was performed by a trained pathologist.

statistical analysis

All data are expressed as mean ± SD. Each in vitro experiment was repeated at least 2-3 times. Three technical replicates were used for all measurements. Statistical analysis was performed using paired and unpaired Student's t-test in Excel 2016. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Example 1

Experimental Example 1 Manufacture of three-dimensional artificial tubular scaffold

3D printing of template, electrospun fiber coating, dip coating of polymer solution, template and particle removal steps were performed for scaffold fabrication. Computer-Aided Design (CAD) based on magnetic resonance imaging (MRI) data was used for the fabrication of the 3D template. Objects of the biliary tree were reconstructed three-dimensionally from 2D magnetic resonance cholangiopancreatography (MRCP) images and post-processed in 3D printable file format (STL) (Fig. 1a). The 3D template was printed with a 3D printer driven by a material compression method on a polyvinyl alcohol filament (PVA filament, ESUN) (Fig. 1b). The open source software Cura was used to generate printable G-codes. The variables of nozzle inner diameter, nozzle temperature, and lamination thickness were set to 0.25 mm, 170 °C, and 0.15 mm, respectively. In order to exclude the surface roughness of the printed template, it was soaked in warm distilled water at 50 °C for 1 minute and sonicated.

For the next step, electrospinning of a polycaprolactone (PCL, MW 80,000; Sigma-Aldrich) solution was performed while rotating the 3D printed PVA template (Fig. 2a). PCL granules were dissolved in a mixture of methylene chloride and dimethylformaldehyde (DMF) in a ratio of 3:1. Solution concentrations were set at 10% and 18% (w/v), respectively, to generate nanoscale and micro, nanoscale mixed electrospinning mats. The two mats were compared for their ability to induce biliary tract cell differentiation prior to template coating. After the comparative experiment, the template coating condition was selected at a concentration of 18% (w/v).

Next, the template on which the fibers were deposited was dip-coated by dipping in a polyurethane solution in which the salt was dispersed, and then the salt particles were leached to form a porous foam layer around the fiber template (Fig. 2b). For the dip coating step, the fiber-coated template was immersed in a solution of thermoplastic polyurethane (TPU; Daelim Chemical) in which salt is dispersed. TPU granules were dissolved in DMF at a concentration of 15% (w/v). Then, the salt particles sieved into a 45 micrometer mesh were mixed with the TPU solution at a concentration of 400% (w/w). After drying the coated part, it was soaked in sonicated water for 2 minutes to filter out salt particles.

The final scaffold having a fiber shape on the surface of the innermost layer was obtained after dissolving the 3D template in distilled water and removing it. A three-dimensional artificial tubular scaffold was obtained by removing the PVA template by soaking it in sonicated water at 50° C. for 30 minutes. During the PVA template removal process, the coated template tip was slightly cut to create an inlet for water penetration.

rabbit artificial bile duct

The shape of the bile duct tree was obtained by processing 3D CAD data from the image of the rabbit body. The final scaffold contained the entire anatomy consisting of two forked intrahepatic conduits (Fig. 3a-a). The layer organization can be observed through the cross-section of the tube wall (Figs. 3a-b). In the step of dip coating on the template on which the fibers were deposited, a fusion region was formed in which the fiber layer and the porous foam layer were combined. This tight fusion ensures no delamination in the layer structure, ensuring adequate mechanical strength for the scaffold. The innermost surface of the tubular scaffold is composed of electrospun fibers (Fig. 3a-c), and the exterior of the scaffold has a porous shape (Fig. 3a-d). Depending on the size of the salt particles used, the pore size is about 50 μm, which can maintain structural flexibility and mechanically support the brittle fibrous layer. The fabricated tubular scaffold maintains the lumen and can withstand physical manipulations occurring during surgery (Fig. 3b). The scaffold was mounted on a tensile tester to demonstrate mechanical stability. As a result, it was confirmed that the fibrous layer and the porous foam layer exhibited greater flexibility with a slightly lower elastic modulus and a much longer elongation at break than the fibrous layer alone (FIG. 3c).

Example 2

Experimental Example 2 hCdH generation and biliary tract cell differentiation

In the production of the three-dimensional artificial tubular scaffold disclosed herein, hCdH-derived biliary tract cells were used. Because primary biliary epithelial cells from humans and other mammals are difficult to isolate and proliferate in vitro, chemically derived hepatic progenitor cells (hCdHs) were utilized for biliary epithelial modeling. To generate hCdH, we recently published (Y. Kim, K. Kang, S. B. Lee, D. Seo, S. Yoon, S. J. Kim, K. Jang, Y. K. Jung, K. G. Lee, V. M. Factor, J. Jeong, D. Choi, J Hepatol 2019, 70, 97.) direct pluripotent progenitor cells from adult human hepatocytes isolated from healthy donor livers via combinatorial treatment with the two small molecules A83-01 and CHIR99021 in the presence of HGF. reprogrammed with When the cells reached passage 2, the biliary tract cell differentiation protocol was applied as shown in Figure 4a. hCdHs cultured for 2 weeks in gelatin-coated dishes in biliary duct cell differentiation medium (CDM) containing Na-Taurocholate and CHIR99021 were identified by immunofluorescence (Fig. 4b) and RT-qPCR analysis as evidenced by the major biliary markers CK7, CK19. , showed significant induction of AE2 and CFTR (Fig. 4c). hCdH-derived biliary tract cells, subsequently referred to as hCdH-Chols, were able to metabolize fluorescein diacetate, consistent with the acquisition of functional maturity (Fig. 5a). Global gene expression profiling confirmed transcriptional changes to the biliary epithelial phenotype during differentiation of hCdHs. The gene expression profile of hCdH-Chols clustered more closely in the human gallbladder than undifferentiated hCdHs (Fig. 5b). These data validate the use of hCdH-Chols for fabrication of functional biliary tract graft constructs by filling tubular scaffolds.

Example 3

Experimental Example 3 Optimization of fibrous layer shape for enhancing biliary tract cell differentiation

Considering the role of cell adhesion and matrix in the regulation of cell behavior, the effect of growth in the fibrous layer on the differentiation properties of hCdH-Chols was tested. For this, hCdH was prepared at the same density (10 ⁶ cells/cm 2 ) in reprogramming medium and plated on standard flat dishes or electrospun fiber scaffolds. After 2 days, the medium was replaced with a biliary duct cell differentiation medium (CDM) containing Na-Taurocholate and CHIR99021 to induce biliary duct cell differentiation for 14 days (Fig. 6a). The results of Live/Dead analysis showed that differences in growth conditions did not affect cell viability. At the end of the experiment, hCdH-Chol plated on the electrospun fibrous layer showed comparable or slightly higher viability than cells cultured in flat dishes (Fig. 6b). In particular, the fibrous layer provided a more favorable microenvironment for biliary cell differentiation. hCdH-Chols cultured in the fibrous layer showed much stronger expression of the major marker genes SSRT2, SCR, JAG1, TGR5, CK7, AE2, CFTR, CK19 and AQP 1 (Fig. 7a). Consistent with this, the growth rate and properties of hCdH-Chols were significantly reduced as judged by RT-qPCR analysis of proliferation (Ki67) and progenitor (CD90, EpCAM) marker gene expression (Fig. 7a). Next, the effect of the fibrous layer structure on the functional properties of hCdH-Chols was evaluated. Given that the concentration and surface tension of the polymer solution define the distribution range of fiber diameters during a typical electrospinning process (MJ Dalby, N. Gadegaard, RO Oreffo, Nat Mater 2014, 13, 558), PCL low and high concentration solutions of (10 and 18% w/v, respectively) on fiber diameter was tested. At low concentrations of PCL, the fiber diameters are narrowly distributed within 1 micrometer (Fig. 8a-a). In contrast, the high concentration polymer solution produced much thicker fibers with a diameter of more than 5 μm at 500 nm, including both nanofibers (red arrow) and microfibers (blue arrow) as shown in FIGS. 8a-b. Mats composed of multi-scale fibers were expected to provide more intimate cell-to-cell and cell-to-matrix interactions due to the enlarged micro-scale pore network compared to sub-micrometer mats (Figs. 8b and c). To demonstrate this point, we observed hCdH and hCdH-Chol cultured in each mat using a scanning electron microscope. When grown on nanofiber-only mats, hCdH did not extend into the fibrous layer and survived on the surface as single cells with minimal cell-to-cell interactions. In contrast, in the multi-scale mat (coarse mat), both hCdHs and specific hCdH-Chols formed significantly larger clusters that were better integrated within the fiber layer network (Fig. 9a). These results were confirmed by RT-qPCR and double immunofluorescence analysis, which confirmed stronger induction of key biliary tract markers and more pronounced expansion of hCdH-Chols in multi-scale mats (rough mats) (Figs. 9b and c). Therefore, the preparation of fibrous layers composed of micro- and nanoscale fibers increased the scaffold porosity, which greatly improved the spatial organization of hCdH and the efficiency of differentiation into biliary tract phenotypes.

Example 4

Experimental Example 4 Epithelialization of artificial biliary tract using hCdH-derived biliary tract cells

Next, we tested whether hCdH could adhere and form a biliary epithelium inside an artificial biliary tract-like structure composed of an inner electrospun fibrous layer and surrounded by a microporous foam layer for increased stability. To this end, hCdH was seeded on tubular scaffolds and cultured for 14 days in a medium promoting biliary duct cell differentiation. Scaffolds from both viewpoints were stained with calcein-AM for 30 min and examined by fluorescence microscopy (Fig. 10a). The results indicated successful epithelialization of the tubular scaffolds. That is, it showed a gradual accumulation of green-calcein fluorescence signal gradually extending over the entire inner surface area (Fig. 10a). In contrast, the cell-free tubular structure used as a negative control did not show any fluorescence signal (Fig. 10a). To evaluate the differentiation status of cells, cross-sections and longitudinal sections of the inner fibrous layer were prepared and stained with antibodies against biliary duct cell-specific proteins CK19 and CFTR. Double immunofluorescence staining showed that the transplanted cells maintained the ability for biliary epithelial differentiation and increased the expression of lineage-specific marker proteins ( FIG. 10b ). In particular, positively stained cells were densely packed and completely colonized on the inner surface of the tubular scaffold (Fig. 10b). Finally, the tubular scaffold was separated and each of the fibrous layer and the porous foam layer was observed through a scanning electron microscope (FIG. 11). As expected, cells were filled only in the inner fibrous layer ( FIG. 11A ) and not in the outer layer ( FIG. 11B ). These data demonstrate that the random mixing of nano and microfibers provides a favorable microenvironment for hCdH attachment, proliferation and differentiation.

Example 5

Experimental Example 5 Evaluation of Substitutability of Natural Biliary Tract

Based on the in vitro results, we investigated the possibility of replacing bioprinted artificial biliary tract structures to restore common bile duct defects in a rabbit model. A 5 mm long segment was excised from a healthy common bile duct to create the defect and for easy tracking, the entire artificial CBD construct filled with hCdH-Chols expressing mCherry (Fig. 12a) was replaced with a 10 mm piece cut out (Fig. 14a). Despite the small wall thickness (440-470 μm, range), the reticulated object had sufficient mechanical strength and flexibility to withstand handling and suturing to both ends of the original common bile duct (Fig. 14b). The rabbit survived the surgery, and from day one moved, ate and behaved normally (Fig. 12b). The experiment was terminated after two weeks as the animals received the immunosuppressive drug Prograf® to alleviate immune rejection of the transplanted human cells. During necropsy, the implanted scaffold remained in place and appeared as an integral part of the rabbit common bile duct ( FIG. 14B ). Despite the yellowing of the lesion site (Fig. 14b), the bile flow through the anastomotic site was maintained as judged by postoperative 3D magnetic resonance cholangiography (MRC) (Fig. 15a). MRC also showed no abnormal bile accumulation other than a slight dilatation of the natural duct at the site of the anastomosis ( FIG. 15A ). Two weeks after surgery, all blood parameters of measured liver function remained within baseline values (Table 1).

Table 1. Rabbit blood test results before and after surgery

However, the levels of bilirubin and aspartate aminotransferase and the number of leukocytes remained elevated 2.7-fold, 1.6-fold, and 2.4-fold, respectively, compared to the preoperative values. These abnormalities are most likely related to slight bile leakage during technically difficult surgeries performed in small sized animals, mild postoperative inflammation, and insufficient healing time. Subsequent histological evaluation of the skull and tail sections of the prepared biliary tract at the anastomosis site showed that the implanted artificial biliary tract construct was well connected to the native rabbit biliary tract (Fig. 13a). Double immunofluorescence staining also confirmed that the mCherry-expressing hCdH-Chol populating the tubular scaffolds expressed the lineage-specific marker proteins CK19 and CK7 ( FIGS. 15b and 13b ). In contrast, none of the two rabbits transplanted with the cell-free 3D printed construct survived more than 5 days. These studies demonstrate the feasibility of ex vivo printed bile duct structures to restore the function of the common biliary tract in vivo.

Claims (9)

In the three-dimensional artificial tubular scaffold,
The three-dimensional artificial tubular scaffold is
an inner fiber layer made of a fibrous biodegradable polymer material;
an outer porous foam layer made of a biocompatible polymer material; and
and a fusion layer in which the biocompatible polymer material is inserted between the fibers of the fiber layer and the two layers are fused;
The fibrous layer comprises one or more cells and two or more fibers having different diameters,
The porous foam layer includes one or more pores,
The fusion layer is a three-dimensional artificial tubular scaffold, characterized in that located between the inner fiber layer and the outer porous foam layer.
According to claim 1,
The fiber layer is a three-dimensional artificial tubular scaffold, characterized in that it comprises a fiber having a diameter of less than 1 micrometer and a fiber of 1 micrometer or more.
According to claim 1,
The cell is a three-dimensional artificial tubular scaffold, characterized in that it comprises hepatic progenitor cells reprogrammed by a medium composition for reprogramming human adult hepatocytes into hepatic progenitors comprising HGF, A83-01 and CHIR99021.
According to claim 1,
The three-dimensional artificial tubular scaffold is a three-dimensional artificial tubular scaffold, characterized in that the artificial biliary tract.
In the three-dimensional artificial tubular scaffold manufacturing method,
The method comprises the steps of
(a) preparing a tubular 3D template using a water-soluble polymer;
(b) electrospinning a biodegradable polymer material on a tubular 3D template in the form of fibers;
(c) immersing the material made in step (b) in a biocompatible polymer solution in which the salt is dispersed;
(d) immersing the material made in step (c) in water after drying; and
(e) seeding the cells in the fibrous layer;
The step (d) is a three-dimensional artificial tubular scaffold manufacturing method, characterized in that it comprises salt leaching through water immersion and removal of the template prepared in step (a).
6. The method of claim 5,
The water-soluble polymer material of step (a) is a three-dimensional artificial tubular scaffold manufacturing method, characterized in that polyvinyl alcohol.
6. The method of claim 5,
3D artificial tubular scaffold manufacturing method, characterized in that the polymer material of step (b) is PCL.
6. The method of claim 5,
The polymer solution of step (c) is a three-dimensional artificial tubular scaffold manufacturing method, characterized in that the polyurethane solution.
6. The method of claim 5,
The cell is a three-dimensional artificial tubular scaffold manufacturing method, characterized in that it comprises hepatic progenitor cells reprogrammed by a medium composition for reprogramming human adult hepatocytes into hepatic progenitor cells comprising HGF, A83-01 and CHIR99021.

Images