KR102656244B1 - Bioreactor - Google Patents

Abstract

Bioreactors are introduced.
For this purpose, the present invention includes a hollow polymer scaffold (100) on which stem cells are implanted on the inner and outer surfaces; Chamber 200; A channel 400 connected to the pump 300 and the cell culture medium 500, and both ends connected to the polymer support; A three-way valve 700 connected to one point of the channel; An air compressor (600) connected to one point of the three-way valve; and a circulation channel 1000 connected to the circulation pump 800 and the liquid composition unit 900, with both ends connected to the chamber.

Description

bioreactor {BIOREACTOR}

The present invention relates to a bioreactor capable of implementing an air-liquid interface (ALI) environment.

Tissue engineering is a field of study that restores biological function by cultivating living tissue or creating a substitute for living tissue and transplanting it. This type of tissue engineering involves culturing the tissue needed in the patient's body in vitro in biodegradable materials for a certain period of time and then transplanting it back into the body. Alternatively, tissue engineering can be done by implanting an artificial scaffold into the body to induce the cells inside the body to proliferate or regenerate.

The artificial scaffold is a structure that can support cells and plays a very important role in cell regeneration. Therefore, it must not only have biocompatible properties by not causing transplant rejection, cytotoxicity, or inflammatory reactions, but also must be efficient in injecting cells and inducing cell proliferation. To facilitate mass transfer, the pore size must be constant, the porosity must be good, the interconnectivity between pores must be excellent, and it must have high mechanical strength to withstand pressure in the living body to serve as a support. .

Artificial scaffolds are mainly manufactured as porous structures using materials such as polylactic acid (PLA) or polyglycolic acid (PGA). These artificial scaffolds are made using various methods to produce polymer porous scaffolds, for example, the solvent-casting/particle-leaching method (Mikos et al. , Polymer, 35, 1068 (1994)), method of expanding polymers using CO2 gas (Gas foaming method: Harris et al., J. Biomed. Mater. Res., 42, 396 (1998)), polymer solution A method of preparing a support by mixing and drying a foaming salt and then foaming the foaming salt in water or an acidic solution (Gas foaming salt method: Nam, et al., J. Biomed. Mater. Res., 53, 1 (2000) ), a method of manufacturing non-woven fabric from polymer fibers (Fiber extrusion and fabric forming process: Paige et al., Tissue Engineering, 1, 97 (1995)), a phase in which the solvent contained in the polymer solution is immersed in a non-solvent to separate the phase Separation method (liquid-liquid phase separation method: Schugens, et al., J. Biomed. Mater. Res., 30, 449 (1996)), in which an emulsified solution mixed with a polymer solution and a non-solvent is rapidly frozen in liquid nitrogen and freeze-dried. Emulsion freeze-drying method (Whang et al., Polymer, 36, 837 (1995)), and piezoelectric spinning method (Electrospinning method: Matthews et al., which produces a fibrous support by directly spinning a polymer solution under an electric field). Biomacromolecules, 3, 232 (2002)), etc.

However, artificial scaffolds manufactured using this method not only have high surface roughness but are also not manufactured with precise structures, despite the fact that the manufacturing process is very complex. In addition, existing artificial scaffolds have the problem of always having errors in biological research using them because it is difficult to control the external/internal shape and it is difficult to reproduce the same shape.

Various research and technological developments are being conducted to solve these problems of existing artificial scaffolds. Among these technologies, a technology called Solid Freeform Fabrication (SFF) is a micro three-dimensional processing technology using rapid prototyping technology and is attracting attention in the field of tissue engineering. Arbitrary shape manufacturing technologies include micro 3D processing technologies such as SLA (stereolithography), FDM (fused depostion modeling), and SLS (selective laser sintering). Artificial scaffolds using arbitrary shape manufacturing technology are manufactured using a machining-based automated manufacturing system, so they can be manufactured in the same standardized shape and can be manufactured as customized scaffolds that take into account the shape of the patient's affected area. In addition, artificial scaffolds using arbitrary shape manufacturing technology can design the internal space to induce blood vessel regeneration, and it is also possible to secure space for regenerating complex tissues or organs.

Recently, in the field of tissue engineering, research is being actively conducted to improve or restore defective tissues and functions of our body by creating artificial structures and implanting them into the body. The development of these artificial structures for tissue transplantation follows the advancement of biotissue engineering, providing functionalized tissue to which cells can attach and be cultured in three dimensions, and is made of biocompatible materials that can coexist with tissues within the body. It must have biodegradable properties and adequate mechanical strength so that the structure can be replaced by cells as cells grow. With the recent development of biotissue engineering, various mechanical engineering techniques are being used to manufacture artificial structures in a more precise and organized manner for the regeneration of human tissue.

Meanwhile, the airway is largely divided into the upper airway and lower airway. The upper airway is made up of the nose, oral cavity, pharynx, and larynx, and the lower airway is divided into the trachea, bronchi, bronchioles, and alveoli. The trachea is the area where the lower respiratory tract begins, and refers to the airway that starts just below the larynx before branching into the main bronchi on both sides. It is shaped like a tube with a diameter of about 2cm, which is the width of your thumb. These organs are composed of cartilage, submucosa, and mucosa layers. Among these, cartilage plays a role in maintaining the shape of the organ, and the submucosa contains glands that secrete mucus into the organ.

Mucosal cells are composed of a columnar epithelium with cilia attached to the top, goblet cells, and basal cells. Among these, cilia play an important role in preventing pneumonia by excreting dust, bacteria, and viruses in the air caught in the mucus secreted by goblet cells to the outside. Additionally, mucosal cells also play a role in supplying moisture to the respiratory tract through various water channels. Mucosal cells are not directly connected to the submucosal layer, but are attached to a membrane called the basement membrane. This is a very essential structure when the mucous membrane is regenerated and is known to be composed of various components.

The frequency of damage to these organs is increasing due to congenital causes, changes in diet, and environmental pollution. The frequency of damage to these organs is also increasing due to reasons such as removal of tumors and damage to organs due to various traumas. In addition, as the aging society increases the frequency of use of artificial respirators, which are essential for the treatment of seriously ill elderly patients, organ damage is emerging as a serious medical problem. To solve this problem, a method of transplanting donor tissue from a recently deceased person into a damaged organ has been reported, but not only is it difficult to obtain donor tissue, but even if donor tissue is obtained, the organ cannot be transplanted until it undergoes a complex preprocessing process over a long period of time. There is a problem.

Recently, attempts have been made to reconstruct organs using artificial structures, but existing artificial scaffolds for airway regeneration have limitations in reproducing the polar structure of tissues after transplantation, and the differentiation efficiency into mucociliaries is low, making it difficult to expel sputum, making it difficult to expel sputum. There remain many problems. Therefore, due to the characteristics of airway mucosal cells, securing an air-liquid interface (ALI) environment is essential for differentiation of ciliated tissue, and in the case of artificial scaffolds, it is possible to simulate the flow of gas-liquid phase exchange in the lumen. There is a need to build an ALI environment.

KR 10-2020-0062236 A

The purpose of the present invention is to develop a bioreactor that can induce alternating flow between gas and liquid phases to induce regeneration and polarity acquisition of the airway mucosa of a polar structure containing cilia on an artificial scaffold for airway regeneration. It is provided.

In order to solve the above problem, the present invention includes a hollow polymer scaffold (100) on which stem cells are implanted on the inner and outer surfaces; Chamber 200; A channel 400 connected to the pump 300 and the cell culture medium 500, and both ends connected to the polymer support; A three-way valve 700 connected to one point of the channel; An air compressor (600) connected to one point of the three-way valve; and a circulation channel 1000 connected to the circulation pump 800 and the liquid composition unit 900, with both ends connected to the chamber.

According to the present invention, it is possible to implement an air-liquid interface (ALI) environment by inducing alternating flow between the gas phase and the liquid phase inside the tubular or tubular support, thereby creating a development and tissue-specific environment. It is possible to simulate the polarity structure of tissues and cells that determine their function, and through this, the characteristic polarity tissue structure of the stomach, intestines, kidneys, pharynx, larynx, respiratory track, blood vessels, organs, or airways. There is an effect that can be copied.

1A and 1B are schematic diagrams of a bioreactor 1 capable of cross-transfer between gas and liquid phases.
Figure 2 is a diagram showing a prototype of a bioreactor implemented based on a schematic diagram of the bioreactor.
Figure 3 is a diagram showing a design and prototype of the chamber 200.
Figure 4 is a diagram confirming the effect of hyaluronic acid-dopamine conjugate coating on the surface of the support over time through the change in intensity measurement of fluorescence labeled with hyaluronic acid.
Figure 5 is a diagram confirming the support coated with fluorescent (fluorosene, FITC) labeled hyaluronic acid-dopamine conjugate:
a and b: 2D fluorescence images of scaffolds coated with fluorescently labeled hyaluronic acid-dopamine conjugate using confocal microscopy (Bar=2 mm);
c: 3D fluorescence image (Bar=500 μm).
Figure 6 is a diagram confirming the effect of MSC transplantation on the surface of the scaffold with or without hyaluronic acid-dopamine conjugate coating using a confocal microscope (Bar=100 μm).
Figure 7 is a diagram evaluating the transplantation ability of MSCs on a scaffold coated with a hyaluronic acid-dopamine conjugate and the MSCs according to the culture time (incubation time) in terms of cell viability.
Figure 8 is a diagram confirming the effect of MSC transplantation inside and outside the d-HA-coated PCL scaffold using 2D and 3D fluorescence images using a confocal microscope (Bar=200 μm).
Figure 9 shows a PCL scaffold with MSCs implanted in a bioreactor capable of alternating liquid and gas phases, and MSCs are differentiated using stem cell cultures with different differentiation medium compositions inside and outside the lumen of the scaffold ( The inside is a diagram confirming the differentiation of stem cells using a differentiation marker (Foxj1 Bar=50 μm / CollagenⅡ Bar=100 μm), airway mucosal epithelial cells (Foxj1), and cartilage (where liquid and gas phases intersect). .

Hereinafter, the present invention will be described in detail through embodiments of the present invention with reference to the attached drawings. However, the following embodiments are provided as examples of the present invention, and if it is judged that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description may be omitted. , the present invention is not limited thereby. The present invention is capable of various modifications and applications within the description of the claims described below and the scope of equivalents interpreted therefrom. The same reference numerals in each drawing indicate members that perform substantially the same function.

In addition, the terminology used in this specification is a term used to appropriately express preferred embodiments of the present invention, and may vary depending on the intention of the user or operator or the customs of the field to which the present invention belongs. Therefore, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part is said to “include” a certain element, this means that it may further include other elements rather than excluding other elements, unless specifically stated to the contrary.

All technical terms used in the present invention, unless otherwise defined, are used with the same meaning as commonly understood by a person skilled in the art in the field related to the present invention. In addition, preferred methods and samples are described in this specification, but similar or equivalent methods are also included in the scope of the present invention. The contents of all publications incorporated by reference herein are hereby incorporated by reference.

The present invention includes a hollow polymer scaffold (100) on which stem cells are implanted on the inner and outer surfaces; Chamber 200; A channel 400 connected to the pump 300 and the cell culture medium 500, and both ends connected to the polymer support; A three-way valve 700 connected to one point of the channel; An air compressor (600) connected to one point of the three-way valve; and a circulation channel 1000 connected to the circulation pump 800 and the liquid composition unit 900, with both ends connected to the chamber.

Hereinafter, preferred embodiments of the bioreactor of the present invention will be described with reference to the drawings.

Figure 1a shows a schematic diagram of the bioreactor 1 of the present invention, and Figure 1b shows a cross section of the polymer support 100 included in the bioreactor of the present invention.

Referring to FIG. 1A, the bioreactor according to an embodiment of the present invention includes a hollow polymer support 100 into which stem cells are implanted on the inner and outer surfaces.

The polymer support may be connected to the channel through a first inlet and a first outlet, which will be described in detail below.

Both ends of the first inlet may be connected to one end of the polymer support and one end of the channel.

Both ends of the first outlet may be connected to the other end of the polymer support and the other end of the channel.

Cell culture medium and sterilized air can be introduced (exited) through the first inflow (outlet) inlet.

The polymer support includes fucoidan, collagen, alginate, chitosan, hyaluronic acid, silk, polyimides, polyamix acid, polycarprolactone, polyetherimide, nylon, polyaramid, polyvinyl alcohol, polyvinylpyrrolidone, polybenzyl-glutamate glutamate, polyphenyleneterephthalamide, polyaniline, polyacrylonitrile, polyethylene oxide, polystyrene, cellulose, polyacrylate, Polymethylmethacrylate, polylactic acid (PLA), polyglycolic acid (PGA), copolymer of polylactic acid and polyglycolic acid (PLGA), poly(ethylene oxide) terephthalate-co -Butylene terephthalate (PEOT/PBT), polyphosphoester (PPE), polyphosphazene (PPA), polyanhydride (PA), polyortho ester (POE), poly A substance selected from the group consisting of (propylene fumarate)-diacrylate (PPF-DA) and poly(ethylene glycol) diacrylate (PEG-DA) It can be manufactured with

Referring to FIG. 1B, the polymer scaffold according to an embodiment of the present invention may be coated on the inner surface (lumen surface) and/or outer surface to enhance stem cell adhesion ability.

In one embodiment, the inner and outer surfaces of the polymer support may be coated with hyaluronic acid. Specifically, the hyaluronic acid is formed by conjugating L-dopamine to the hyaluronic acid chain 111 to form a catechol group 112. It may be hyaluronic acid-dopamine conjugate (d-HA) (110).

The hyaluronic acid can be attached to the polymer support by the adhesive catechol group 112 attached to the hyaluronic acid chain 111.

The polymer support may have a tubular structure with a lumen.

The polymer support can be used for trachea regeneration or respiratory track regeneration.

The polymer support may include a polarized tissue structure including cilia on the polymer support.

In the interior (lumen) of the polymer support, sterilized air and cell culture fluid can interact to create an air-liquid interface (ALI) environment.

Figure 3 shows a schematic diagram of the chamber 200 of the present invention.

Referring to Figures 3 and 1A, the bioreactor according to an embodiment of the present invention includes a chamber 200.

The shape of the chamber 200 is not limited, and it can be applied to the present invention as long as part or all of the polymer support can be wrapped from the outside.

Inside the chamber 200, a composition capable of inducing differentiation of stem cells into specific cells, including cartilage cells, may be contained.

In one implementation, the chamber may include a first inlet 210 and a first outlet 220.

In another embodiment, the chamber may include plugs 230 and 240 connected to one (other) side of the chamber.

The plug may be connected to both sides of the chamber to separate the interior and exterior spaces of the chamber.

In another embodiment, the chamber may include an insert 201 consisting of a second inlet 250, a second outlet 260, and a shaft 270.

The chamber may be connected to a circulation channel, which will be described in detail below, through a second inlet and a second outlet.

Both ends of the second inlet may be connected to one side of the chamber and one end of the circulation channel.

Both ends of the second outlet may be connected to the other side of the chamber and the other end of the circulation channel.

The liquid composition 910 may be introduced (exited) through the second inlet (exit) inlet.

The polymer support can be fixed by the shaft.

Also, referring to FIG. 1A, the bioreactor according to an embodiment of the present invention includes a channel 400.

Both ends of the channel may be connected to a polymer support.

The channel may be connected to the pump 300, the cell culture medium 500, and/or the three-way valve 700.

The cell culture medium 510 may be introduced (exited) into or out of the polymer support by the pump.

The cell culture medium can be continuously circulated by the pump.

The cell culture fluid portion may contain cell culture fluid so that the cell culture fluid can be introduced and/or withdrawn along the channel.

In one embodiment, the three-way valve may include a cell culture fluid inlet 710, a sterile air inlet 720, and an outlet 730.

The three-way valve may have an opening that can be selectively opened and closed, and its opening and closing operation may be controlled by a separately provided control unit (not shown).

In another implementation, the three-way valve may be connected to the air compressor 600.

In another implementation, the air compressor may include a filter 620.

The air compressor may generate sterilized air 610 from external air 630 through a filter.

Sterile air may be introduced (exited) into (out) the polymer support by the air compressor.

Sterile air can be continuously circulated by the air compressor.

In summary, by selectively opening and closing the three-way valve, the cell culture fluid introduced from the cell culture portion and the sterilized air introduced from the air compressor can be alternately and repeatedly introduced (exited) into and out of the polymer support.

Also, referring to FIG. 1A, the bioreactor according to an embodiment of the present invention includes a circulation channel 1000.

Both ends of the circulation channel may be connected to the chamber.

The circulation channel may be connected to the circulation pump 800 and/or the liquid composition unit 900.

The liquid composition 910 may be introduced into (out of) the chamber by the circulation pump.

The liquid composition can be continuously circulated by the circulation pump.

The liquid composition portion may support the liquid composition so that the liquid composition can be drawn in and/or withdrawn along the circulation channel.

In one embodiment, the liquid composition allows stem cells transplanted to the polymer scaffold to differentiate into airway mucosal epithelial cells (120).

In another embodiment, the liquid composition allows stem cells transplanted to the polymer scaffold to differentiate into chondrocytes.

In one embodiment, the bioreactor may be for reproducing the structure of polarized tissue, and the polarized tissue may be epithelium tissue or respiratory epithelium tissue.

In one embodiment, the bioreactor is capable of differentiating stem cells transplanted inside and outside the polymer support into different cells.

In one embodiment, the bioreactor differentiates the inside of the polymer support by alternately passing a liquid phase (cell culture medium) and a gas phase (sterile air) having a composition of a respiratory epithelium differentiation medium, and the outside contains chondrocytes. ) Differentiation can be achieved by culturing in a liquid phase (cell culture medium) with a differentiation medium composition.

In one embodiment, the bioreactor can control the shear stress delivered to cells by controlling the speed of cross-flow of the gas phase and liquid phase within the polymer support.

In one embodiment, stem cells can be transplanted into a polymer scaffold by culturing (incubating) the polymer scaffold in a culture medium containing stem cells for 24 to 96 hours.

In one embodiment, stem cells can be transplanted into the polymer scaffold by culturing the polymer scaffold in a culture medium containing 5Х10 ⁴ cell/ml to 5Х10 ⁶ cell/ml for 24 to 96 hours.

In one embodiment, the stem cells may be adult stem cells, more preferably mesenchymal stem cells (MSCs), umbilical cord mesenchymal stem cells (UCMSCs), or adipose-derived It may be adipose derived mesenchymal stem cell (ADMSC) or bone marrow-derived mesenchymal stem cell (BMMSC).

In the present invention, the term “stem cell” refers to an undifferentiated cell having the ability to self-replicate and differentiate and proliferate. Stem cells include subpopulations such as pluripotent stem cells, multipotent stem cells, and unipotent stem cells, depending on their differentiation ability.

The pluripotent stem cells refer to cells that have the ability to differentiate into all tissues or cells that make up a living body, and multipotent stem cells have the ability to differentiate into multiple types of tissues or cells, although not all types. refers to cells that have Unipotent stem cells refer to cells that have the ability to differentiate into specific tissues or cells.

Pluripotent stem cells include embryonic stem cells (ES Cells), undifferentiated germline cells (EG Cells), and pluripotent stem cells (iPS cells). Pluripotent stem cells include mesenchymal stem cells (adipose-derived, Examples include adult stem cells such as bone marrow (derived from bone marrow, cord blood or umbilical cord derived, etc.), hematopoietic stem cells (derived from bone marrow or peripheral blood, etc.), nervous system stem cells, and reproductive stem cells. Unipotent stem cells usually do not divide. Examples include committed stem cells, which exist in a state of low activity and then actively divide to produce only liver cells.

In one embodiment of the present invention, mesenchymal stem cells were typically used.

Any suitable method for isolating and collecting stem cells can be used. For example, induced pluripotent stem cells typically enter a pluripotent state from somatic cells that have been “reprogrammed” by ectopic expression of transcription factors such as Oct4, Sox2, Klf4, c-MYC, Nanog, and Lin28. [Reference: Takahashi et al., Cell 131:861-72 (2007); Park et al., Nature 451:141-146 (2008); Yu et al., Science 318:1917-20 (2007)]. Cord blood stem cells can be isolated from fresh or frozen umbilical cord blood. Mesenchymal stem cells can be isolated, for example, from raw, unpurified bone marrow or ficoll-purified bone marrow. In some cases, flow cytometry-based methods (e.g., fluorescenceactivated cell sorting) can be used to sort cells based on the presence or absence of specific cell surface markers. there is. If non-autologous cells are used, consideration should be given to the selection of immune type-matched cells so that the organ or tissue will not reject the organ or tissue when transplanted into the subject. Isolated cells can be washed with a buffered solution (e.g., phosphate-buffered saline) and resuspended in cell culture medium. Standard cell culture methods can be used to culture or expand cell populations.

In one aspect, the present invention includes the steps of manufacturing a polymer support by electrospinning or 3D printing a polymer material; and a method for producing a polymer scaffold including the step of transplanting stem cells inside and outside the polymer scaffold.

In one aspect, the present invention relates to a polymer support with a tubular structure prepared by the method of the present invention.

In one embodiment, the polymer support may be used for regeneration of the stomach, intestines, kidneys, pharynx, larynx, blood vessels, or trachea, and is more preferably used for regeneration of trachea.

In one aspect, the present invention relates to a polymer support for regenerating a tubular respiratory track (respiratory track) manufactured by the method of the present invention.

In one aspect, the present invention includes the steps of mounting the polymer support having a tubular structure of the present invention into the bioreactor of the present invention; And the inside of the polymer scaffold is differentiated by alternately passing through a liquid phase and a gas phase having a differentiation medium composition for respiratory epithelial cells, and the outside is cultured in a liquid phase having a chondrocyte differentiation medium composition for differentiation. It relates to a method of manufacturing a prosthetic organ (Trachea) support.

In one aspect, the present invention includes the steps of mounting the polymer support having a tubular structure of the present invention into the bioreactor of the present invention; And the inside of the polymer scaffold is differentiated by alternately passing through a liquid phase and a gas phase having a differentiation medium composition for respiratory epithelial cells, and the outside is cultured in a liquid phase having a chondrocyte differentiation medium composition for differentiation. It relates to a method of manufacturing an artificial respiratory track support.

In one aspect, the present invention relates to a prosthetic organ (Trachea) support manufactured by the manufacturing method of the present invention.

In one embodiment, respiratory epithelial cells on the scaffold may include a polar structure containing cilia.

In one aspect, the present invention relates to an artificial respiratory track support manufactured by the manufacturing method of the present invention.

In one embodiment, the support may include a polar structure including cilia.

Using the bioreactor of the present invention, the composition of bioactive substances contained in the cell culture medium inside and outside the polymer support is adjusted, and the internal stem cells are differentiated into mucous membranes by creating an air-liquid interface environment through cross-flow between the liquid and gas phases. It is possible to simulate the characteristic polarized tissue structure of an organ or airway by inducing differentiation of external stem cells into hyaline cartilage.

In the present invention, the cell culture medium provided to the bioreactor may include a liquid phase with a differentiation medium composition, and depending on the target cell for differentiation, appropriate growth factors, for example, vascular endothelial growth factor (VEGF) , TGF-β growth factor, bone morphogenetic proteins (e.g., BMP-1, BMP-4), platelet derived growth factor (PDGF), basic fibroblast growth factor (b- FGF), such as FGF-10, insulin-like growth factor (IGF), epidermal growth factor (EGF), or growth differentiation factor-5 (GDF) -5) etc. [Reference: Desai and Cardoso, Respir. Res. 3:2 (2002)].

The present invention will be described in more detail through the following examples. However, the following examples are only for illustrating the content of the present invention and are not intended to limit the present invention.

Example 1. Fabrication of a bioreactor capable of alternating liquid and gas phases

A bioreactor for manufacturing cell scaffolds that mimics the polarized tissue structure of organs in the human body was manufactured. Specifically, cross-flow is performed by alternating gas and liquid phases so that efficient mucociliary differentiation of stem cells can be induced on the luminal surface of a cell scaffold that mimics a trachea with a polar structure containing cilia among polar tissue structures. A bioreactor connected to a pump was manufactured to control the speed of phase transfer while inducing an alternating flow to create an air-liquid interface (ALI) environment (FIGS. 1 to 3).

Example 2. Fabrication of a scaffold implanted with MSCs for organ simulation

2-1. Fabrication of a PCL scaffold coated with hyaluronic acid-dopamine conjugate (d-HA) in which L-dopamine is substituted for hyaluronic acid

To confirm the coating effect of the hyaluronic acid-dopamine conjugate on the surface of the scaffold, d-HA (hyaluronic acid-dopamine conjugate with L-dopamine substituted for hyaluronic acid) labeled with fluorescent fluorescent particles (FITC) (d-HA) The PCL support was added to a PBS buffer solution (pH 8.5) in which -FITC) was dissolved and stored at 37°C to perform surface coating. To measure the degree of coating, the scaffold was collected at specific times (12, 18, 24, 48, and 72 hours), and then the d-HA-FITC-coated PCL scaffold was washed three times with a PBS solution, and the hyaluronic acid was decomposed. Shiki treated hyaluronidase and measured the fluorescence intensity of fluorocein (FITC), which was separated by decomposition of hyaluronic acid on the surface of the PCL support. As a result, it was confirmed that the intensity of fluorescence increased with time, and did not increase after 48 hours (Figure 4). Through this, it was confirmed that the most appropriate time for coating d-HA on the PCL support was 48 hours, and it was inferred that this PCL support coating was formed through the adhesive ability of L-dopamine conjugated to hyaluronic acid.

In addition, after 48 hours, the d-HA-FITC-coated scaffold was confirmed using a confocal microscope (LSM510, Carl Zeiss, Germany), and as a result, green fluorocein (FITC) was present throughout the inside and outside of the scaffold. Since it was evenly distributed, it was confirmed that d-HA could effectively and evenly coat the surface of the PCL support (Figure 5).

2-2. Establishment of MSC transplantation conditions onto d-HA coated PCL scaffolds

To confirm the effect of transplantation of mesenchymal stem cells (MSC) (expressing CD44, a hyaluronic acid receptor) onto the PCL scaffold depending on whether or not the hyaluronic acid-dopamine conjugate was coated, the PCL scaffold coated with the hyaluronic acid-dopamine conjugate was tested. The scaffold and the uncoated PCL scaffold were each stored in cell culture containing 5Х10 ⁵ cells/ml of MSC, and after 48 hours, the scaffold was stained with DAPI for the nuclei (blue), and the nuclei were stained using a confocal microscope. Cells were confirmed. As a result, it appeared that MSCs were transplanted inside and outside of the hyaluronic acid-coated PCL scaffold, whereas MSCs were not observed in the PCL scaffold that was not coated with hyaluronic acid (Figure 6). It is inferred that the effective transplantation effect of MSCs onto the PCL scaffold is through specific binding between the hyaluronic acid on the surface of the PCL scaffold and the hyaluronic acid receptor (CD44) of the MSCs.

In addition, in order to confirm the transplantation efficiency of MSCs onto the hyaluronic acid-coated PCL scaffold according to the culture time, the hyaluronic acid-coated PCL scaffold was added to a cell culture medium containing 5Х10 ⁵ cells/ml of MSC, stirred, and cultured. , the scaffold was collected at specific times (3, 6, 12, 24, 48, 72, and 96 hours), washed three times with PBS solution, and cell viability was checked using MTT assay to determine the amount of MSCs transplanted to the scaffold. Measured. As a result, it was confirmed that the amount of MSCs transplanted to the scaffold increased with time, and did not increase after 72 hours (Figure 7). In addition, the scaffold collected after 72 hours was stored in a cell culture medium containing 5Х10 ⁵ cells/ml of MSC for an additional 48 hours, but it was confirmed that the amount of transplanted MSC did not increase. Through this, it was confirmed that the most appropriate culture time for transplanting MSCs onto the surface of a scaffold coated with hyaluronic acid was 48 hours.

2-3. Fabrication of d-HA coated PCL scaffolds implanted with MSCs

MSCs were transplanted onto the 3D printed PCL scaffold under the d-HA coating conditions and MSC transplantation conditions optimized in Examples 2-1 and 2-2. After transplantation, the nuclei were stained with blue DAPI and confirmed in 3D images using a confocal microscope. As a result, it was confirmed that MSCs were effectively transplanted inside and outside the d-HA-coated PCL scaffold (Figure 8).

Example 3. Confirmation of differentiation of scaffold into airway epithelial cells using bioreactor

The PCL support on which the MSCs prepared in Example 2 were transplanted was installed in a bioreactor capable of providing alternating liquid and gas phases prepared in Example 1. Through the bioreactor, the inside of the scaffold was alternately passed through a liquid phase and a gas phase containing a mucosal epithelial cell differentiation medium composition, and the outside of the scaffold was stored in a liquid phase containing a chondrocyte differentiation medium composition. After 48 hours, the degree of differentiation inside and outside of the MSC-transplanted scaffold using the bioreactor and the medium for inducing stem cell differentiation into airway mucosal epithelial cells and chondrocytes was confirmed through immunostaining. As a result, it was confirmed that MSCs inside and outside the scaffold expressed FOXJ1, a mucosal epithelial cell differentiation marker, and CollageII, a chondrocyte differentiation marker, respectively (Figure 9), through which MSCs inside the scaffold differentiated into mucosal epithelial cells. It was confirmed that the MSCs outside the scaffold were differentiated into osteocytes, producing an artificial scaffold that mimics the tracheal mucosa, which is a polar tissue.

1: Bioreactor
100: polymer support 110: hyaluronic acid-dopamine conjugate
111: Hyaluronic acid chain 112: Catechol group
120: Airway mucosal epithelial cells
200: Chamber 201: Insert
210: first inlet 220: first outlet
230, 240: plug 250: second inlet
260: second outlet 270: shaft
300: pump 400: channel
500: Cell culture fluid part 510: Cell culture fluid
600: Air compressor 610: Sterile air
620: Filter 630: Outside air
700: three-way valve 710: cell culture fluid inlet
720: Sterile air inlet 730: Outlet
800: Circulation pump 900: Liquid composition section
910: Liquid composition 1000: Circulation channel

Claims (12)

A hollow polymer scaffold (100) with stem cells implanted on its inner and outer surfaces;
Chamber 200;
A channel 400 connected to the pump 300 and the cell culture medium 500, and both ends connected to the polymer support;
A three-way valve 700 connected to one point of the channel;
An air compressor (600) connected to one point of the three-way valve; and
A bioreactor (1) comprising a circulation channel (1000) connected to a circulation pump (800) and a liquid composition unit (900), with both ends connected to the chamber:
The bioreactor is capable of creating an air-liquid interface (ALI) environment inside the polymer support by alternating between sterilized air and cell culture fluid by selectively opening and closing the three-way valve 700. The method of claim 1, wherein the chamber
First inlet 210;
First outlet (220);
Plugs (230, 240) connected to one (other) side of the chamber; and
Insert 201 consisting of a second inlet 250, a second outlet 260, and a shaft 270;
A bioreactor comprising: According to clause 1,
A bioreactor wherein the polymer support and the channel are connected by a first inlet and a first outlet. According to clause 1,
A bioreactor wherein the chamber and the circulation channel are connected by a second inlet and a second outlet. According to clause 1,
The polymer support includes fucoidan, collagen, alginate, chitosan, hyaluronic acid, silk, polyimides, polyamix acid, polycarprolactone, polyetherimide, nylon, polyaramid, polyvinyl alcohol, polyvinylpyrrolidone, polybenzyl-glutamate glutamate, polyphenyleneterephthalamide, polyaniline, polyacrylonitrile, polyethyleneoxide, polystyrene, cellulose, polyacrylate, poly Methyl methacrylate, polylactic acid (PLA), polyglycolic acid (PGA), copolymer of polylactic acid and polyglycolic acid (PLGA), poly(ethylene oxide) terephthalate-co- Butylene terephthalate (PEOT/PBT), polyphosphoester (PPE), polyphosphazene (PPA), polyanhydride (PA), polyorthoester (POE), poly(propylene) A material selected from the group consisting of poly(propylene fumarate)-diacrylate (PPF-DA), and poly(ethylene glycol)diacrylate (PEG-DA). A bioreactor is manufactured. According to clause 1,
A bioreactor in which hyaluronic acid is coated on the inner and outer surfaces of the polymer support. According to clause 6,
The bioreactor wherein the hyaluronic acid is hyaluronic acid (d-HA) conjugated with L-dopamine. According to clause 1,
The polymer support is a bioreactor that is cultured for 24 to 96 hours in a culture medium containing stem cells of 5Х10 ⁴ cell/ml to 5Х10 ⁶ cell/ml. According to clause 1,
The polymer support is a bioreactor for trachea regeneration or respiratory track regeneration. According to clause 1,
The bioreactor wherein the polymer support includes a polarized tissue structure including cilia on the polymer support. According to clause 1,
The stem cells are mesenchymal stem cells, bioreactor.
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