KR102504479B1 - 3D artificial tubular-scaffold and manufacturing method thereof - Google Patents

Abstract

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

Description

3D artificial tubular-scaffold and manufacturing method thereof

The disclosure herein relates to a three-dimensional artificial tubular scaffold that can be implanted 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 prepare artificial scaffolds, implant them into living bodies, and replace the functions of defective organs or organs in living bodies with the transplanted scaffolds.

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

In addition, research is being conducted to enable artificial scaffolds to perform biological functions in addition to physical functions that replace the functions of defective organs or organs. Specifically, research is being conducted to seed cells capable of exhibiting the biological characteristics of an organ on an artificial scaffold so that the cells can perform biological functions while differentiating and proliferating within the artificial scaffold.

Disclosed herein is an artificial scaffold having mechanical strength and flexibility suitable for in vivo transplantation, an artificial scaffold suitable for cell attachment, proliferation and differentiation, and a manufacturing method thereof in the field of artificial scaffold research as described above. it's about

delete

Republic of Korea Patent Publication No. 10-2009-0004027

An object of the present specification is to provide a three-dimensional artificial tubular scaffold characterized by 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 including: The artificial tubular scaffold includes an inner fibrous layer made of a fibrous biodegradable polymer material; A porous foam layer made of a biocompatible polymeric material; and a fusion layer in which the biocompatible polymeric material is inserted between the fibers of the fibrous layer and the two layers are fused, wherein the fibrous layer includes at least one cell and at least two fibers having different diameters, and the porous foam layer comprises It includes one or more pores, and the fusion layer is characterized in that it is located between the inner fibrous layer and the outer porous foam layer.

In one embodiment, the fibrous layer provides a three-dimensional artificial tubular scaffold, characterized in that it includes both fibers having a diameter of less than 1 micrometer and fibers having a diameter of 1 micrometer or more.

In one embodiment, the cells are a three-dimensional artificial tubular scan comprising hepatic progenitor cells reprogrammed by a reprogramming medium composition of human adult hepatocytes to hepatic progenitor cells including HGF, A83-01 and CHIR99021. provide a fold.

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 method for manufacturing a three-dimensional artificial tubular scaffold comprising the following steps.

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

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

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

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

In one embodiment, the method for manufacturing the three-dimensional artificial tubular scaffold includes liver progenitor cells reprogrammed by a reprogramming medium composition of human adult hepatocytes containing HGF, A83-01 and CHIR99021. It is characterized by doing.

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

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

Problems with the prior art

In conventional artificial scaffolds for in vivo implantation, many studies have been conducted to prepare scaffolds using biocompatible polymeric materials. Conventional artificial scaffolds are prepared by preparing a biocompatible polymer solution and electrospinning to form a layer in the form of fibers. However, there was a problem in that the fiber shape was easily broken and did not have sufficient mechanical strength required for the transplant operation process.

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

Lastly, for artificial scaffolds containing conventional cells, many studies have been conducted to attach cells to the artificial scaffold. However, conventional scaffolds have been focused on finding “specific” fiber diameters or pore sizes that are optimized for cell adhesion. However, the diameter or pore size of a specific fiber is limited to providing 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, conventional scaffolds have a problem in that cells penetrate into and adhere to the space within the scaffold structure through various spaces and are intensively attached to the surface of the scaffold. This problem has a limitation in not having proper interactions between cells and cells and between cells and matrices.

The present specification discloses a three-dimensional artificial tubular scaffold and a manufacturing method thereof to solve the above problems.

Construction of a three-dimensional artificial tubular scaffold

In general, in the case of a tubular scaffold for in vivo implantation, it must have appropriate flexibility to withstand pressure due to the flow of a solution and prevent damage to the scaffold due to movement after implantation in vivo. In addition, it should have adequate mechanical strength to prevent tearing caused by sutures during transplant 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 appropriate flexibility, mechanical strength, and an appropriate environment in which cells can perform biological functions. The three-dimensional artificial tubular scaffold includes a fiber 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 part 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 fiber layer includes fibers having nanoscale diameters and fibers having microscale diameters. In a preferred embodiment, the fibrous layer includes both fibers having a diameter of less than 1 μm and fibers having a diameter of 1 μm or more. This fiber configuration provides more variable 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 herein has spaces of various sizes, cells penetrate more deeply into the fibrous layer and are attached thereto.

8-9, it can be seen that cells survive on the surface in the fibrous layer composed of only nanoscale fibers, but cells form a cell population in the scaffold and survive in the fibrous layer in which both nanoscale and microscale fibers exist. there is. In addition, due to these characteristics, interactions between cells and cells and between cells and matrices may be improved, and the differentiation efficiency of cells may be improved.

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

The porous foam layer is located on the outermost side 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 flexibility due to the pores present in the porous foam layer. Depending on the size of the pores, the mechanical strength and flexibility of the scaffold may be affected. At this time, the size of the pore can be adjusted to have mechanical strength and flexibility suitable for in vivo transplantation.

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

The three-dimensional artificial tubular scaffold disclosed herein is characterized in that a porous foam layer is present in addition to the fibrous layer. Typically, scaffolds containing only fibrous layers do not have adequate mechanical strength for artificial tubular scaffolds for implantation, because fibers tend to break and become brittle. 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 serves to structurally support the broken and brittle fibrous layer to increase the mechanical strength of the scaffold. 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 an 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 the fibrous layer. According to FIG. 3c, it can be seen that the case composed of the fiber layer and the porous foam layer exhibits greater flexibility with a lower modulus of elasticity and long elongation at break than the case composed only of the fiber 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 exists in a form in which a 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 herein has an effect of increasing mechanical strength and flexibility as the fibrous layer and the porous foam layer exist together, and also has an effect of preventing peeling between the two layers. Simply stacking each layer for scaffold manufacturing may cause peeling of each layer because the bonding between the layers is not strong. However, the three-dimensional artificial tubular scaffold disclosed herein has a fusion layer in which a fibrous layer and a porous foam layer are fused, so that the bonding between the layers is strong, so that it can function as a single scaffold without separation of each layer.

Three-dimensional artificial tubular scaffold materials

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

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

Since the porous foam layer of the scaffold disclosed herein must continuously perform physical functions after being implanted in vivo, it must not self-decompose or disappear 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 implantation in vivo, the porous foam layer is made of a biocompatible polymer material. The biocompatible, non-degradable polymeric 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, and polyurethanes can be more than one. However, it is not limited thereto. One example 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 perform physical as well as biological functions when implanted in a living body.

The cells may be cells of a transplant target and foreign cells, allogeneic cells, or autologous cells. In one embodiment, the cells may be autologous cells 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 perform functions in vivo after being transplanted to a transplant target.

The cells may be stem cells or differentiated cells. 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 depending on the transplantation site of the scaffold. In one embodiment, the cells are embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells, placental stem cells, dedifferentiated stem cells, endothelial cells, epithelial cells, skin cells, endoderm cells, mesenchymal 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, it is not limited thereto. One example is a biliary tract cell. One example is liver progenitor cells.

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

Functions of 3D artificial tubular scaffolds

Performing physical functions in vivo

The three-dimensional artificial tubular scaffold disclosed herein can perform physical functions by replacing defective organs in vivo. The three-dimensional artificial tubular scaffold disclosed in this specification can be implanted into a living body due to its appropriate mechanical strength and flexibility and function to maintain a normal flow of materials in the living body. 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 and transplanted into a living body. Cells attached to the artificial scaffold perform biological functions 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 provides an environment for attachment, proliferation, and differentiation of cells to perform a function of tissue regeneration in vivo. In one embodiment, the three-dimensional artificial tubular scaffold disclosed herein may perform a function of regenerating bile duct tissue.

Uses of three-dimensional artificial tubular scaffolds

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

therapeutic use

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

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

Experiment use

As an example, 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 into a non-human animal, and the implanted non-human animal may be compared with an unimplanted non-human animal to evaluate the transplantation effect.

Characteristics of the 3D artificial tubular scaffold

First, the three-dimensional artificial tubular scaffold disclosed herein has a fiber 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, 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 making the scaffold thick 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 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 a fiber layer and a porous foam layer. The fusion layer exists in a form in which a biocompatible polymer material constituting the porous foam layer is inserted between the fibers of the fibrous layer. The fusion layer serves to strengthen the bond between the fiber layer and the porous foam layer. The fusion layer serves to prevent separation between the fibrous layer and the porous foam layer so that the layer structure of the scaffold can function as one scaffold without being separated.

Thirdly, 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 diameters of the fibers constituting the fibrous layer are diversified. The fiber layer containing fibers of different diameters serves to vary the size of the space within the fiber layer. Spaces within the fibrous layer serve to allow cells to freely penetrate and adhere to the fibrous layer structure. Spaces within the fibrous layer serve 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 the fibrous layer to perform biological functions.

The three-dimensional artificial tubular scaffold disclosed herein includes one or more cells in a fibrous layer. The cells can proliferate and differentiate by being attached to the fibrous layer in the scaffold. The cells can perform biological functions after the scaffold is implanted in vivo. The cells can function to regenerate tissues in vivo after the scaffold is implanted in vivo.

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.

As an example, the method

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

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

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

(d) immersing the material made in step (c) 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 on the fibrous layer after step (d).

Each step is described in detail below.

Manufacturing a tubular 3D template with a water-soluble polymer material

3D templates can be fabricated from water-soluble polymeric materials to prepare scaffold structures suitable for implantation.

The 3D template may be manufactured using a mold suitable for a transplanted organ or structure of the organ, and then manufactured using the same, or may be manufactured according to a 3D printing method. In one 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 one embodiment, the 3D template may be manufactured according to a 3D printing method using magnetic resonance image data (MRI). In one 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 plate stacking method, or a directional energy deposition method. In one 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 manufacturing 3D templates include polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), polyethylene oxide (PEO), carboxyl methyl cellulose (CMC), starch, polyacrylic acid (PAA), and hyaluronic acid (PAA). Hyaluronic acid) may be any one or more selected from the group consisting of. However, it is not limited thereto. In one embodiment, the water-soluble polymer material is polyvinyl alcohol.

An appropriate 3D printer nozzle inner diameter, nozzle temperature, and layer thickness can be set for 3D template manufacturing. In one embodiment, the nozzle inner diameter, the nozzle temperature, and the layer thickness are about 0.1 to 0.4 mm, 150 to 190 ° C, and 0.05 to 0.25 mm, respectively. In one embodiment, the nozzle inner diameter, the nozzle temperature, and the layer 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, nozzle temperature, and laminate thickness are about 0.25 mm, 170° C., and 0.15 mm, respectively.

Surface treatment steps for 3D templates

The step of smoothing the surface of the prepared 3D template may be selectively performed. Physical or chemical methods may be used to smooth the surface of the 3D template. In order to smooth the surface of the 3D template, mechanical processing, ultrasonic processing, laser processing, electric discharge processing, electrolytic processing, etc. may be used. In one embodiment, an ultrasonic machining method is used to smooth the surface of the 3D template.

For example, in order to smooth the surface of the 3D template, the template may be immersed in distilled water and then subjected to ultrasonic treatment. At this time, the distilled water is about 40 ~ 60 ℃ in a specific embodiment, the distilled water is about 50 ℃. In addition, the time to immerse the template in distilled water is about 30 seconds to 2 minutes. In one embodiment, the time to immerse 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 the biodegradable polymer material in a “fiber form” on the surface of the 3D template, the biodegradable polymer material must be manufactured in the form of a fiber. Various spinning methods may be used to prepare biodegradable polymeric materials in the form of fibers. In one embodiment, an electrospinning method may be used to prepare a biodegradable polymer material in a fiber form.

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

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, it is not limited thereto. The suitable solvent may be one or more substances among 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.

Biodegradable polymer materials 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 may be adjusted to deposit the biodegradable polymer material in the form of fibers having various diameters on the surface of the 3D template. 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). A preferred embodiment is 18% (w/v).

Step of immersing the 3D template on which fibers are deposited into a biocompatible polymer solution

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

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

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

The biocompatible polymer solution is prepared by dissolving a biocompatible polymer material in an appropriate solvent. The biocompatible polymeric material is at least one selected from the group consisting of polyethylene, polypropylene, polytetrafluoroethylene, poly(ethylene terephthalate), poly(methyl methacrylate), poly(2-hydroxyethyl methacrylate), poly(N-isopropylacrylamide), polysiloxane, and polyurethanes. can However, it is not limited thereto. The suitable solvent may be one or more substances among 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 to 25% (w/v). In one embodiment, the concentration of the solution is about 10 to 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 the property of being leached by a solvent into the biocompatible polymer solution. In one embodiment the solvent is water. In one embodiment, the salt may be any one or more salts having a property of being leached by reacting with water. However, it is not limited thereto. In one embodiment the salt is NaCl. In one embodiment, a 400% (w/w) NaCl solution was used as a salt-dispersed biocompatible polymer solution.

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 can 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 about 30 micrometers. In one embodiment, the salt particle size is greater than about 40 micrometers. In one embodiment, the salt particle size is greater than 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 a salt particle sieved to a mesh of about 40 to 50 micrometers. In one embodiment, the salt particles are salt particles sieved to about 45 micrometer mesh.

Drying the template immersed in a salt-dispersed biocompatible polymer solution and then immersing in water

The template immersed in the salt-dispersed biocompatible polymer solution is then immersed in water after drying. In one embodiment, the template may be immersed in water to leach salt particles. In one embodiment, the template may be immersed in water for about 1 minute or more to leach salt particles. In a preferred embodiment, the template may be immersed in water for about 2 minutes to leach out the salt particles. In one embodiment, the water may be sonicated water. Through this process, a porous foam layer including pores may be formed in the template.

The template immersed in the salt-dispersed biocompatible polymer solution is dried and then immersed in water so that the first 3D template manufactured using the water-soluble polymer material can be removed. In one embodiment, after being immersed in a biocompatible polymer solution in which salt is dispersed, the dried template may be immersed in water for about 10 minutes or longer. In one embodiment, the template may be immersed in water for about 20 minutes or longer. 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 to 70 ° C. 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 one embodiment, the water may be sonicated water.

The process of drying the template immersed in a biocompatible polymer solution in which salt is dispersed, leaching the salt by immersing it in water, and removing the first 3D template manufactured using a water-soluble polymer material may occur together or in a sequential order. can In one embodiment, salt leaching through the water immersion step and a process of removing the first 3D template using a water-soluble polymer material may occur simultaneously. In one embodiment, after salt leaching through the water immersion step, the first 3D template manufactured using a water-soluble polymer material may be removed.

A step of cutting the end of the template before or in the middle of the step of immersing the template in water after drying the template immersed in the biocompatible polymer solution in which the salt is dispersed may be additionally included. When the end of the template is cut and then immersed in water, the first 3D template manufactured using a water-soluble polymer material can be more effectively removed.

Preparatory steps before seeding cells on the fibrous layer

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

Seeding cells into the fibrous layer

The method for manufacturing a three-dimensional artificial tubular scaffold disclosed herein may include drying a template dipped in a salt-dispersed biocompatible polymer solution and then immersing the template in water, followed by seeding the cells. The cells may be seeded on a fibrous layer in an artificial tubular scaffold.

The cells may be cells derived from the same species as the cells of the transplantation target, or cells derived from a different species. In one embodiment, the cells may be cells 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, dedifferentiated stem cells, endothelial cells, epithelial cells, skin cells, endoderm cells, mesenchymal 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, it is not limited thereto. One example is liver progenitor cells. One example is a biliary tract cell. One embodiment is liver progenitor cells reprogrammed from adult human hepatocytes. One embodiment is biliary tract cells differentiated from hepatic progenitor cells reprogrammed adult human hepatocytes. One embodiment is hepatic progenitor cells reprogrammed by a medium composition for reprogramming human adult hepatocytes to hepatic progenitor cells comprising HGF, A83-01 and CHIR99021.

Use of 3D artificial tubular scaffold manufacturing method

The method for manufacturing a three-dimensional artificial tubular scaffold disclosed herein may be used for preparing an organ to be transplanted into a living body or a scaffold customized for an organ. However, it is not limited thereto and may be used for other suitable purposes.

The three-dimensional artificial tubular scaffold disclosed herein can be used to prepare a scaffold suitable for an organ or organ size and structure suitable for in vivo transplantation. In the method for manufacturing a three-dimensional artificial tubular scaffold disclosed herein, a 3D template of a size and structure customized for transplantation can be manufactured through 3D design using magnetic resonance images of organs or organs to be transplanted and 3D printing using the same. A three-dimensional artificial tubular scaffold having a transplant-specific size structure can be manufactured using the prepared 3D template.

The three-dimensional artificial tubular scaffold disclosed herein may include a step of seeding cells suitable for a transplant target and an organ or organ to be transplanted. In one embodiment, an artificial tubular scaffold including cells derived from a transplant target adapts quickly after being transplanted to a transplant target and can perform its function in vivo. In one embodiment, an artificial tubular scaffold including organs or organ-specific cells to be transplanted can be implanted in vivo and perform biological functions according to the transplanted organ or organs.

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 skilled in the art that the scope of the present invention is not limited by these examples.

Experiment method

MRI imaging

MRI was performed 1 day before and 14 days after surgery using a 3T MRI scanner (Ingenia; Philips Healthcare, Best, The Netherlands) with a small distal coil. Rabbits were anesthetized with an intramuscular injection of ketamine (40-100 mg/kg) and xylazine (5-13 mg/kg) during MRI scans. Briefly, three-plane localization imaging gradient echo sequences were first obtained, followed by breath-evoked 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; Materialise, Louvain, Belgium). .

Biliary cell differentiation

This experiment was performed according to the protocol approved by the Institutional Review Board of Hanyang University (HYI-16-229-3). Human liver tissue was collected from three donors who underwent surgery at Hanyang University Medical Center with the consent of the patients. The production of hCdH is 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 changed to DMEM/F12 (Gibco, CA, USA) reprogramming medium (then HAC called) was changed. hCdHs were generated 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 administered with DMEM/F12 supplemented with 7.5ng/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. were seeded on gelatin (Sigma) coated dishes in biliary cell differentiation medium (CDM) containing

immunofluorescence staining

Cells, scaffolds and biliary tracts 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). PCR cycles were 20 seconds at 95 ° C and 40 seconds at 60 ° C, for a total of 40 cycles.

list of primers

Fluorescein diacetate (FD) assay

Cells were washed with PBS and incubated with fluorescein diacetate (Sigma) diluted in medium at a concentration of 2.5 μg/mL for 15 minutes at 37°C. After removing the FD containing medium, the cells were cultured 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

Media was removed and 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 fragments were incubated at 37 °C. and incubated for 30 minutes at 5% CO2. The medium was then removed and replaced with fresh medium. After that, pictures were taken using a fluorescence microscope.

cell seeding

Prior to cell seeding, the fibrous scaffold and artificial biliary tract were cut, washed with ethanol and sterilized with UV light. After 30 minutes, the fibrous scaffold and artificial biliary tract were coated with 0.1% gelatin (Sigma) and incubated at 37 °C and 5% CO2 for 30 minutes. Excess gelatin was then removed and samples were dried at room temperature for 2 h. hCdH was subcultured using previously established methods. 10 6 cells were suspended in 10 μl of HAC medium and seeded inside the artificial bile duct construct. Then incubated for one hour at 37 °C and 5% CO2. In a 4-well plate, 0.5 ml of reprogramming medium was used per well and replaced with CDM medium the next day.

SEM imaging

The microstructures of the electrospun fibers and cultured cells were visualized using scanning electron microscopy. The scaffolds with cells were washed three times with PBS and fixed in a 5% glutaraldehyde solution containing 0.1 M sodium cacodylate and 0.1 M sucrose for 30 minutes. After washing with distilled water for 5 min, the sample was slowly dehydrated by successive incubation in 50%, 60%, 70%, 80%, 90%, and 100% ethanol solutions for 5 min each, followed by hexamethyldisilazane (HMDS; JT Baker, Phillipsburg, NJ; USA) for 15 minutes. Once completely dry, the sample was sputtered with Pt until a 20 nm thick coating was obtained. Samples were imaged through an 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 an 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 the Illumina TruSeq Stranded mRNA Sample Prep Kit (Illumina, Inc., San Diego, CA, USA, # RS-122-2101). An early step in library preparation involved purifying poly-A containing mRNA molecules using magnetic beads attached to poly-T. After this step, the purified mRNA was fragmented into small pieces using divalent cations at elevated temperature. The truncated mRNA fragment was copied onto 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 were subjected to a process of end repair by adding a single 'A' base and an adapter linkage. After this step, a final cDNA library was generated and amplified by PCR. The resulting library was quantified using the KAPA Library Quantification Kit for the Illumina Sequencing Platform according to the qPCR Quantification Protocol Guide (KAPA Biosystems, # KK4854) and verified by a TapeStation D1000 ScreenTape (Agilent Technologies, # 5067-5582). Indexed libraries were paired-end sequenced by Macrogen Inc.'s Illumina Hiseq2500 (Illumina, Inc., San Diego, CA, USA).

Cryosectioning and H&E staining

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

Bioinformatics analysis

The Illumina standard pipeline and real-time analysis (RTA) tool uses BCL2Fastq (v2.20, Illumina Inc., San Diego, CA, USA) from paired-end RNA sequencing data for raw image processing, base calling and It was used to generate FASTQ data. The 101bpX2 read sequence was preprocessed using Sickle (v1.33) (32) to trim bad subsequences or CutAdapt (v1.5) (33) to remove adapter sequences. After pre-processing, sequencing reads were read from the Hg19 human reference genome (University of California, University of California iGenomes, https://support. Available at illumina.com/sequencing/sequencing_software/igenome.html). The amount of expression level of a transcript is measured by the TPM (Transcripts Per Kilobase Million) score, making it easy to compare the proportion of reads mapped to genes in each sample. Raw sequencing data are available from GEO (https://www.ncbi.nlm.nih.gov/geo) under 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).

implantation in vivo

Animal experiments were performed in compliance with NIH guidelines and Hanyang University animal research protocols with the permission of the Hanyang University IACUC (Institutional Animal Care and Use Committee No. 2018-0097A). 21-month-old female semi-specific-pathogen-free rabbits weighing 3-4 kg were provided from Duyeol Biotech (Seoul, Republic of Korea). All animals were housed under standard conditions with a 12-h light/dark cycle and fed standard chow during the 4-week acclimatization period. Rabbits were fasted for 12 hours before surgery. Under general anesthesia administered by an anesthesiologist, the animals were fixed in a supine position and a laparotomy was performed through a midline incision in the upper abdomen to expose the common bile duct (CBD). The lower CBD was incised and the artificial biliary tract was anastomized to the proximal and distal ends of the CBD with 10-0 Ethilon® disconnect sutures under light microscopy. All procedures were performed by a microsurgeon. Adequate biliary drainage through the artificial biliary tract anastomosed intraoperatively was confirmed. 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 3D Artificial Tubular Scaffold

3D printing of the template, electrospun fiber coating, dip coating of the polymer solution, and 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 three-dimensionally reconstructed from 2D magnetic resonance cholangiopancreatography (MRCP) images and post-processed into a 3D printable file format (STL) (Fig. 1a). The 3D template was printed with a 3D printer driven by a material compression method using a polyvinyl alcohol filament (ESUN) (Fig. 1b). The open-source software Cura was used to generate G-code for printing. The variables of nozzle inner diameter, nozzle temperature and layer thickness were set to 0.25 mm, 170 °C and 0.15 mm, respectively. In order to remove the surface roughness of the printed template, it was immersed in warm distilled water at 50 °C for 1 minute and sonicated.

For the next step, electrospinning of 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. The solution concentration was set at 10% and 18% (w/v), respectively, to create nanoscale and micro, nanoscale mixed electrospun mats. The two mats were compared for their ability to induce biliary cell differentiation prior to template coating. After the comparative experiment, the template coating condition was selected as 18% (w/v) concentration.

Next, the template on which the fibers were deposited was immersed in a polyurethane solution in which salt was dispersed, and then dip-coated, and 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 salt-dispersed thermoplastic polyurethane (TPU; Daelim Chemical) solution. TPU granules were dissolved in DMF at a concentration of 15% (w/v). Then, the salt particles sieved to a 45 micron mesh were mixed with the TPU solution at a concentration of 400% (w/w). After drying the coated part, it was immersed in sonicated water for 2 minutes to filter out salt particles.

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

Rabbit artificial biliary tract manufacturing

The shape of the biliary tree was obtained by processing 3D CAD data from images of the rabbit body. The final scaffold contained the entire anatomy consisting of two bifurcated intrahepatic ducts (Fig. 3a-a). Layer organization can be observed through cross-sections of the tube walls (FIGS. 3a-b). In the step of dip-coating the fiber-deposited template, a fusion region where the fiber layer and the porous foam layer are combined is formed. This tight fusion prevents delamination of the layered structure, ensuring adequate mechanical strength for the scaffold. The innermost surface of the tubular scaffold was composed of electrospun fibers (Fig. 3a-c), and the outer surface of the scaffold was porous (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 fragile fibrous layer. The fabricated tubular scaffold maintains a lumen and can withstand physical manipulations occurring during surgery (Fig. 3b). Scaffolds were mounted in a tensile tester to demonstrate mechanical stability. As a result, it was confirmed that the fiber layer and the porous foam layer exhibited greater flexibility with a slightly lower modulus of elasticity and a much longer elongation at break than the fiber layer alone (FIG. 3c).

Example 2

Experimental Example 2 hCdH production and biliary cell differentiation

In the preparation of the three-dimensional artificial tubular scaffold disclosed herein, hCdH-derived biliary cells were used. Because primary biliary epithelial cells from humans and other mammals are difficult to isolate and propagate in vitro, chemically derived hepatic progenitor cells (hCdHs) were utilized for biliary epithelial modeling. To generate hCdH, we used recent publications (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.) Directly converting adult human hepatocytes isolated from healthy donor livers into bipotent progenitor cells by combinatorial treatment with the two small molecules A83-01 and CHIR99021 in the presence of HGF as described in (2019, 70, 97). reprogrammed with When the cells reached passage 2, the biliary cell differentiation protocol was applied as shown in Figure 4a. hCdHs cultured for 2 weeks on gelatin-coated dishes in biliary cell differentiation medium (CDM) containing Na-Taurocholate and CHIR99021 expressed the major biliary markers CK7, CK19, as demonstrated by immunofluorescence (Figure 4b) and RT-qPCR analysis. , showed marked induction of AE2 and CFTR (FIG. 4c). hCdH-derived biliary cells, hereafter 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 a biliary epithelial phenotype during differentiation of hCdHs. The gene expression profile of hCdH-Chols clustered closer to human gallbladder than undifferentiated hCdHs (Fig. 5b). These data validate the use of hCdH-Chols for the fabrication of functional biliary graft constructs by filling tubular scaffolds.

Example 3

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

Given the role of cell adhesion and matrix in the regulation of cell behavior, the effect of growth on the fibrous layer on the differentiation properties of hCdH-Chols was tested. To this end, 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 biliary cell differentiation medium (CDM) containing Na-Taurocholate and CHIR99021 to induce biliary 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 electrospun fibrous layers showed comparable or slightly higher viability than cells cultured on flat dishes (Fig. 6b). In particular, the fibrous layer provided a more favorable microenvironment for biliary cell differentiation. hCdH-Chols cultured on the fibrous layer showed much stronger expression of the key marker genes SSRT2, SCR, JAG1, TGR5, CK7, AE2, CFTR, CK19 and AQP 1 (Fig. 7a). Consistent with this, the growth rate and characteristics of hCdH-Chols were significantly reduced as judged by RT-qPCR analysis of proliferative (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 during a typical electrospinning process define the range of fiber diameter distribution (MJ Dalby, N. Gadegaard, RO Orefo, 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 comparison, high-concentration polymer solutions produced much thicker fibers ranging from 500 nm to more than 5 μm in diameter, including both nanofibers (red arrows) and microfibers (blue arrows), as shown in Fig. 8a-b. Mats composed of multiscale fibers were expected to provide more intimate cell-to-cell and cell-to-matrix interactions due to the enlarged microscale pore network compared to submicrometer mats (Figures 8b and c). To demonstrate this point, we observed hCdH and hCdH-Chol cultured on 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 multi-scale mats (rough mats), both hCdHs and specific hCdH-Chols formed significantly larger clusters that were better integrated within the fibrous 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 prominent expansion of hCdH-Chols in multiscale mats (rough mats) (Figure 9b and c). Therefore, the fabrication of the fibrous layer composed of micro- and nanoscale fibers greatly improved the spatial organization of hCdH and the differentiation efficiency into the biliary phenotype by increasing the scaffold porosity.

Example 4

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

Next, we tested whether hCdH could adhere and form biliary epithelium inside an artificial biliary-like structure composed of an inner electrospun fibrous layer and surrounded by a microporous foam layer to increase stability. To this end, the tubular scaffold was seeded with hCdH and cultured for 14 days in a medium promoting biliary cell differentiation. At both points, scaffolds were stained with Calcein-AM for 30 min and examined under a fluorescence microscope (FIG. 10A). Results indicated successful epithelialization of the tubular scaffold. That is, it showed a gradual accumulation of green-calcein fluorescence signal that gradually expanded over the entire inner surface area (Fig. 10a). In contrast, the cell-free tubular construct used as a negative control did not show any fluorescence signal (FIG. 10a). To evaluate the differentiation status of the cells, transverse and longitudinal sections of the inner fibrous layer were prepared and stained with antibodies against the biliary cell-specific proteins CK19 and CFTR. Double immunofluorescence staining showed that the transplanted cells retained the capacity for biliary epithelial differentiation and increased expression of lineage specific marker proteins (FIG. 10B). In particular, positively stained cells were densely packed and fully colonized on the inner surface of the tubular scaffold (FIG. 10B). Finally, the tubular scaffold was separated and each of the fiber layer and the porous foam layer was observed through a scanning electron microscope (FIG. 11). As expected, cells were populated only in the inner fibrous layer (FIG. 11A) and absent in the outer layer (FIG. 11B). These data prove 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 replacement possibility of natural bile duct

Based on the in vitro results, the possibility of replacing bioprinted artificial bile duct constructs to restore common bile duct defects in a rabbit model was investigated. A 5 mm long segment was excised from a healthy common bile duct to create a defect, and the entire artificial CBD construct (FIG. 12A) filled with mCherry expressing hCdH-Chols was replaced with a cut 10 mm piece (FIG. 14A). Despite their small wall thickness (ranging from 440 to 470 μm), the mesh-like objects possessed sufficient mechanical strength and flexibility to withstand handling and suturing of both ends of the original common bile duct (Fig. 14b). Rabbits survived the surgery and moved, ate and behaved normally from day one (Fig. 12b). The experiment was terminated after 2 weeks as the animals received the immunosuppressive drug Prograf® to mitigate immune rejection of transplanted human cells. During necropsy, the implanted scaffold remained in place and appeared to be an integral part of the rabbit common bile duct (FIG. 14B). Despite yellowing of the lesion site (FIG. 14B), bile flow through the anastomosis 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 conduits at the site of the anastomosis (FIG. 15A). Two weeks after surgery, all measured hematological parameters of 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 leukocyte counts remained elevated 2.7-fold, 1.6-fold, and 2.4-fold, respectively, compared to preoperative values. These abnormalities are most likely related to slight bile leakage, mild postoperative inflammation, and insufficient healing time during technically challenging surgeries performed in small-sized animals. Subsequent histological evaluation of the cranial and caudal sections of the biliary tract prepared at the site of the anastomosis 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, neither of the two rabbits implanted with the cell-free 3D printed construct survived longer than 5 days. These studies demonstrate the feasibility of bioprinted bile duct constructs to restore the function of the common bile duct in vivo.

Claims (9)

In the three-dimensional artificial tubular scaffold,
The three-dimensional artificial tubular scaffold
An inner fibrous layer made of a fibrous biodegradable polymer material;
An external porous foam layer made of a biocompatible polymeric 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,
The fibrous layer includes 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 fibrous layer and the outer porous foam layer.
According to claim 1,
The fibrous layer is a three-dimensional artificial tubular scaffold, characterized in that it comprises fibers with a diameter of less than 1 micrometer and fibers with a diameter of 1 micrometer or more.
According to claim 1,
The three-dimensional artificial tubular scaffold, characterized in that the cells include hepatic progenitor cells reprogrammed by a reprogramming medium composition of human adult hepatocytes to hepatic progenitor cells including 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 method of manufacturing the three-dimensional artificial tubular scaffold of claim 1,
The method includes the following steps,
(a) preparing a tubular 3D template using a water-soluble polymer;
(b) electrospinning a biodegradable polymer material in the form of fibers onto a tubular 3D template;
(c) immersing the material made in step (b) in a biocompatible polymer solution in which salt is dispersed;
(d) immersing the material made in step (c) in water after drying; and
(e) seeding the cells into the fibrous layer;
The method of manufacturing the three-dimensional artificial tubular scaffold of claim 1, wherein step (d) comprises salt leaching through water immersion and removal of the template prepared in step (a).
According to claim 5,
The method for manufacturing a three-dimensional artificial tubular scaffold according to claim 1, wherein the water-soluble polymer material in step (a) is polyvinyl alcohol.
According to claim 5,
The method of manufacturing the three-dimensional artificial tubular scaffold of claim 1, wherein the polymer material in step (b) is PCL.
According to claim 5,
The method of manufacturing the three-dimensional artificial tubular scaffold of claim 1, wherein the polymer solution in step (c) is a polyurethane solution.
According to claim 5,
The cells are prepared from the three-dimensional artificial tubular scaffold of claim 1, characterized in that they contain hepatic progenitor cells reprogrammed by a reprogramming medium composition of human adult hepatocytes into hepatic progenitor cells containing HGF, A83-01 and CHIR99021. method.

Images