KR102126733B1 - Method of preparing a structure consisted of arranged fiber structure and cells - Google Patents

Abstract

The present invention relates to a manufacturing method for fabricating a structure including an arrayed fiber/surface structure and arrayed cells using a three-dimensional cell printing technique. The manufacturing method of the present invention enables the fabrication of cell-carrying collagen fiber structures arranged in one direction, thereby obtaining a more effectively and efficiently arranged cell structure. The structure is expected to be applied to various fields related to the regeneration of organs or arranged tissues by enabling a faster cell growth and cell differentiation effect than that of a generally produced collagen structure.

Description

Method of preparing a structure consisted of arranged fiber structure and cells}

The present invention relates to a manufacturing method for fabricating a structure including an arrayed fiber/surface structure and an arrayed cell using a three-dimensional cell printing technique.

Tissue engineering is an area of collaboration between several disciplines that seek to develop biological products that regenerate, maintain, or enhance the functioning of tissues using principles of life sciences and engineering. A typical method is a procedure of artificially forming tissue by separating and culturing cells from tissues to be regenerated, and inoculating them with appropriate biomaterials to amplify and culture them.

These procedures require a suitable cell support that is easy to deliver cells to the required areas, and to make new tissues that can function as mechanical aids and function as a three-dimensional structure for tissue growth. These supports must have an appropriate microstructure that allows cells to proliferate and create a unique matrix, and have many pores interconnected in three dimensions, so that the cells can grow inside, and provide the nutrients needed for cell growth. It should be able to supply. In addition, it should be a biodegradable material that is non-toxic and can be completely degraded and disappeared in vivo after termination of its function as a support.

To improve the performance of artificial tissues, recent artificial tissues have cell printing technology due to the fact that they can retain growth factors and cells in a desired location, distribute cells uniformly, and effectively carry growth factors. techniques) are widely applied in various tissue regeneration fields. For successful tissue regeneration, the cell-mounted carrier must have high porosity and pore size control, oxygen and nutrient supply, and 100% interconnection between pores for angiogenesis.

On the other hand, cells must be cultured and differentiated in an arranged form in order to regenerate tissue having an arranged structure. Therefore, many research teams have tried to produce cell supports having a fibrous structure arranged in various ways to cultivate cells in an arrayed form. However, processes that are frequently used to fabricate arrayed fiber structures, such as electrospinning processes and lithography processes, cannot be stacked, so they have limitations in being used as cell carriers, so much research is needed.

Republic of Korea Application Publication No. 10-2017-0012099

The present inventors have repeatedly studied the structure for tissue regeneration, and the collagen structure carrying cells of the arranged structure is recognized by the fact that cells must be cultured and differentiated in the arranged form in order to regenerate the tissue having the arranged structure. Invented the manufacturing method for production, and experimentally confirm that the collagen structure carrying cells of the arranged structure through the above manufacturing method has a faster cell growth and cell differentiation effect than the produced collagen structure. Completed.

Accordingly, the object of the present invention is (a) myoblast (myoblast), Preparing a bioink containing one or more cells and collagen selected from the group consisting of cardiomyoblast, smooth muscle cell, neural cell, and vascular cells step;

(b) arranging the collagen and progenitor cells in one direction by applying a shear stress to the bioink of step (a);

(c) dispensing the bioink of step (b) onto a plate to form an artificial structure; And

(d) It provides a method for producing a cell support, comprising the step of maintaining the curing and alignment of the artificial structure of step (c) using a potassium chloride-glycine solution.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems that are not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention as described above, the present invention (a) myoblast (myoblast), Preparing a bioink containing one or more cells and collagen selected from the group consisting of cardiomyoblast, smooth muscle cell, neural cell, and vascular cells step;

(b) arranging the collagen and progenitor cells in one direction by applying a shear stress to the bioink of step (a);

(c) dispensing the bioink of step (b) onto a plate to form an artificial structure; And

(d) providing a method of manufacturing a cell support, comprising the step of maintaining the curing and alignment of the artificial structure of step (c) using a potassium chloride-glycine solution.

In one embodiment of the present invention, the collagen in step (a) may be included in 3 to 9% by weight based on the total weight of the bioink.

In another embodiment of the present invention, the number of progenitor cells in step (a) may be 1 x 10 ⁶ cells/mL ^-1 to 1 x 10 ⁹ cells/mL ^-1 .

In another embodiment of the present invention, the temperature of the plate in step (c) may be 33°C to 40°C.

In another embodiment of the present invention, the distribution of step (c) may be made at a flow rate of 0.7 uLs ^-1 to 0.9 uLs ^-1 .

In another embodiment of the present invention, the distribution of step (c) may be performed at a speed of 9 mms ^-1 to 11 mms ^-1 .

In another embodiment of the present invention, the distribution of step (c) may be made such that the artificial structure forms a lattice structure.

In another embodiment of the present invention, in the potassium chloride-glycine solution of step (d), the potassium chloride may be 50 to 200 mM and the glycine may be 50 to 100 mM.

The method of manufacturing an arrayed collagen fiber structure and a structure having cells of the present invention enables production of a cell-supported collagen fiber structure arranged in one direction, and more effectively and efficiently arranged cell structures can be obtained, and the structure is generally produced. It is expected to be applied to various fields related to arranged tissue or organ regeneration because it has a fast cell growth and cell differentiation effect compared to collagen structure.

1 shows a schematic diagram of constructing a structure having arranged collagen fiber structures and cells.
Figure 2 shows the morphology of a structure with arranged fibrous structures and cells.
Figure 3 is a comparison of the shape of the fabric structure prepared according to the flow rate of bio ink.
Figure 4 is a comparison of the shape of the collagen fiber structure according to the movement speed during bioprinting.
5a to 5g show the results of various cell experiments and the culture of the construct containing the progenitor cells.

The present inventors have repeatedly studied the structure for tissue regeneration, and the collagen structure carrying cells of the arranged structure is recognized by the fact that cells must be cultured and differentiated in the arranged form in order to regenerate the tissue having the arranged structure. Invented the manufacturing method for production, and experimentally confirm that the collagen structure carrying cells of the arranged structure through the above manufacturing method has a faster cell growth and cell differentiation effect than the produced collagen structure. Completed.

In one embodiment of the present invention, it was confirmed that it is possible to manufacture a structure having fibers and cells arranged according to the manufacturing method of the present invention (see Example 2).

In another embodiment of the present invention, the optimal flow rate was confirmed by observing the shape of the fiber structure according to the flow rate of the bioink (see Example 3).

In another embodiment of the present invention, the optimum movement speed was confirmed by observing the shape of the fiber structure according to the movement speed during bioprinting (see Example 4).

In another embodiment of the present invention, as a result of performing the cell array culture and cell experiment, the cell growth rate was similar, but it was confirmed that the cell array differentiation was superior to the cell array. (See Example 5).

Hereinafter, the present invention will be described in detail.

The present invention (a) myoblast (myoblast), Preparing a bioink containing one or more cells and collagen selected from the group consisting of cardiomyoblast, smooth muscle cell, neural cell, and vascular cells step;

(b) arranging the collagen and progenitor cells in one direction by applying a shear stress to the bioink of step (a);

(c) dispensing the bioink of step (b) onto a plate to form an artificial structure; And

(d) providing a method of manufacturing a cell support, comprising the step of maintaining the curing and alignment of the artificial structure of step (c) using a potassium chloride-glycine solution.

The term “collagen” used in the present invention is a light protein that is mainly present in the bones, skin, and muscles of animals and is also distributed in cartilage, organ membranes, hair, etc., and is a natural biocompatible material. In the present invention, collagen may be used in place of other natural biocompatible materials such as gelatin, fucoidan, alginate, chitosan or hyaluronic acid.

In the present invention, the (a) collagen may be preferably included in 3 to 9% by weight relative to the total weight of the bioink. This is because when collagen is contained in less than 3% by weight relative to the total weight of bioink, printing in a 3D shape is impossible, and as the weight ratio of collagen increases, the structure is difficult to produce due to the high viscosity and the survival rate of the cells inside the ink also decreases. .

The term “muscular cell” used in the present invention refers to muscle cells in an undifferentiated state, and when differentiation of progenitor cells progresses, it fuses with other cells to form multinucleated elongated myotubes and muscle fibers. ). The effect of promoting differentiation of progenitor cells according to the manufacturing method of the present invention may be derived from skeletal muscle, myocardium and smooth muscle, etc., and the progenitor cells may be cardiomyoblasts. In addition, in addition to the progenitor cells, smooth muscle cells, neural cells, and vascular cells may be used for bioink production, but are not limited thereto.

The term “potassium chloride-glycine solution” used in the present invention helps to form the fibers of collagen, and thereby helps to maintain the arrangement of collagen fibers. In the potassium chloride-glycine solution of step (d), the potassium chloride may be 50 to 200 mM and the glycine may be 50 to 100 mM, but is not limited thereto.

The potassium chloride-glycine solution may have optimal efficiency when used with collagen.

The number of source cells of step (a) in the present invention is 1 x 10 ⁶ cells / mL ^-1 to 1 x 10 ⁹ cells / mL ^-1 days, but not limited ^{to, 1 x 10 6 cells / mL} - ¹ or more is sufficient.

In the present invention, the temperature of the plate in step (c) is 33°C to 40°C It may be, but is not limited to, collagen is effectively gelated in the temperature range.

In the present invention, the distribution of step (c) may be made at a flow rate of 0.7 uLs ^-1 to 0.9 uLs ^-1 , but is not limited thereto. However, at a flow rate of 0.9 or more, the alignment may disappear, and at a flow rate of 0.7 or less, unstable discharge may cause a problem that the thickness of the strands is not uniform.

The distribution of step (c) in the present invention may be made at a speed of 9mm s ^-1 to 11mm s ^-1 , but is not limited now. However, the alignment may be lost at a speed of 9 mm s ^-1 or less, and the alignment may be maintained at a speed of 12 mm s ^-1 or more, but a problem may appear instability in the production of strands.

In the present invention, the distribution of step (c) may be made such that the artificial structure forms a lattice structure. This is to provide pores to the structure, which plays an essential role in cell survival, proliferation, and migration. If the pore connectivity of the structure is low, mass transport and exchange of nutrients do not occur well, which in turn leads to cell necrosis in large three-dimensional structures. Is caused.

Hereinafter, preferred embodiments are provided to help understanding of the present invention. However, the following examples are only provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

Example 1. Preparation of bio ink

Preparation of the bio-ink is as shown in Figure 1, the bio-ink was prepared by mixing C2C12 cells (1 X 10 ⁷ cells mL ^-1 ) in a 5 wt% neutral collagen solution.

Example 2. Preparation of structure with arranged fibrous structure and cells

The method of manufacturing the structure having the arranged fibrous structure and cells is as shown in FIG. 1. In the case of bioink, C2C12 cells (1 X 10 ⁷ cells mL ^-1 ) were mixed in a 5 wt% neutral collagen solution, and the mixed solution as described above was 0.80 uL s ^-1 flow rate and 10 mm in a 37°C plate. After printing at a rate of s ^-1 , it was reacted with 150 mM KCL / 50 mM L-glycine solution (pH 7.5). Then, the structure prepared as above was observed as follows through optical photography, scanning electron micrograph and fluorescence photography.

In order to confirm the arrangement of cells, Live (green) / Dead (red) staining was performed to stain live and dead cells. The structure was stained for 1 hour in 0.15 mM cacein AM (live) and 2 mM ethidium homodimer-1 (dead), and then measured. In addition, the structure was reacted with phalloidin conjugated with Alexa Fluor 488 (Invitrogen, USA) solution for 2 hours or more to confirm the alignment of collagen fibers, and then measured.

As a result, as shown in Figures 1 and 2, in the case of the structure produced by the above method, it was confirmed that the cells and collagen fibers have alignment.

Example 3. Observation of the shape of the fiber structure according to the flow rate of bioink

Collagen structures were prepared under the conditions of 0.64 to 0.96 uL s ^-1 (fixed at a rate of 10 mm s ^-1 ) to observe the fiber structure different from the flow rate of bioink. All conditions except the flow rate of collagen were performed in the same manner, and the alignment was observed using a scanning electron microscope, and the angle of the fiber structure on the scanning electron microscope photograph was measured to calculate the full width at half maximum (FWHM).

As a result, as shown in FIG. 3, it was confirmed that the alignment disappeared at a flow rate of 0.9 uL s ^-1 or higher, and the thickness of the strand was not uniform due to unstable discharge at a flow rate of 0.7 uL s ^-1 or lower.

Example 4. Observation of the shape of the fiber structure according to the movement speed during bio printing

Collagen structures were prepared under the conditions of 5 to 12.5 mm s ^-1 to observe the fiber structure different from the flow rate of bioink. When printing, all conditions except the moving speed were the same, and alignment was confirmed by scanning electron microscopy and collagen fluorescence staining. The half width was calculated by measuring the angle of the fiber structure on the scanning electron microscope.

As a result, as shown in FIG. 4, it was confirmed that the alignment was lost at a speed of 9 mm s ⁻¹ or less, and the alignment was maintained at a speed of 12 mm s ⁻¹ or more, but it was confirmed that it was unstable during the production of the strand.

Example 5. Structure culture and cell experiment with progenitor cells

In order to confirm the cellular capacity of the prepared cell-collagen structure (CS-AF) having the arrayability, a comparison was made with the structure (CON) lacking the arrayability without using potassium chloride-glycine buffer under the same printing conditions.

5-1. Cell culture

DMEM (Sigma-Aldrich, DMEM) containing collagen construct (12 × 8 × 1 mm3) containing C2C12 cells, the progenitor cells, and FBS (Gemini Bio-Products, USA) and 1% antibiotic (Antimycotic; Cellgro, Manassas, VA) USA) cultured in a medium at 37° C. under 5% CO ₂ conditions. The medium was changed once every 2 days, and DMEM medium containing 2% horse serum and 1% antibiotic was used to induce differentiation of myoblasts.

5-2. Cell proliferation test

Each cell construct was cultured for 1, 3, 7 days and then subjected to MTT assay using a cell proliferation kit (Cell Proliferation Kit I; Boehringer Mannheim, Germay). The cultured construct was 0.5 mg mL ^-1 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (3-(4,5-dimethylthiazol-2-yl) After reacting with -2,5-diphenyltetrazolium bromide (MTT) for 4 hours, it was measured using a microplate reader with an absorbance of 570 nm.

As a result, it was confirmed that the difference in cell proliferation was not observed in the two constructs as shown in FIG. 5A.

5-3. Fluorescence image acquisition method

Collagen constructs containing each cell were cultured for 3 days and 7 days, stained by treating with 0.15 mM calcein AM and 2 mM ethidium homodimer-1 at 37°C for 30 minutes, and living cells (green) and dead cells (red). Was measured with a fluorescence microscope.

As a result, as shown in FIG. 5B, both structures showed high cell viability in fluorescence pictures stained with live and dead cells, but in the CS-AF on the 7th day, unlike the CON, the structure in which the cells were arranged was confirmed.

In addition, in order to obtain the myosin heavy chain (MHC) and sarcomeric alpha actinin (α-actinin) images of cells cultured in each collagen construct, washed twice with PBS after 7 and 14 days of culture, 3.7% The paraformaldehyde was treated and fixed at room temperature. And again, the structure was washed twice with PBS, immersed in 2% Triton X-100 for 60 minutes, and then treated with 2% BSA for 120 minutes, and the sample was diluted 1:50 with anti-MHC. Antibodies (Invitrogen, USA) and anti-α-actinin antibodies (Invitrogen) were stored at 4° C. overnight. The secondary antibody conjugated with Alexa Fluor 488 (1:50, Molecular Probes) for MHC and Alexa Fluor 594 (1:50, Molecular Probes) for α-actinin was treated at room temperature for 1 hour, and 5 μM diamidino- 2-phenylindole (diamidino-2-phenylindole, DAPI; Invitrogen, Carlsbad, CA) and phalloidin 15 U/mL (Invitrogen, Carlsbad, CA) were conjugated to Alexa Fluor 568 for control staining. Images were acquired using a Zeiss confocal microscope, and ImageJ software was used to calculate cell orientation (cell orientation).

As a result, when synthesizing the staining pictures and cell alignment results shown in FIGS. 5C to 5F, it was confirmed that CS-AF shows high alignment.

5-4. Real-time polymerase chain reaction (RT-PCR)

RT-PCR (Real-time polymerase chain reaction) was performed after 7, 14 and 21 days of culture to quantitatively measure the expression level of Myogenic markers Myod1, Myof5, Myogin, and Myh2. Total RNA for use in RT-PCR was separated using TRI reagent (Sigma-Aldrich), and the purity and concentration of the isolated RNA was measured by a spectrophotometer (FLX800T; Biotek, USA). CDNA was synthesized from RNase-free DNase-treated total RNA (500 ng) using a reverse transcription system, and qRT with StepOnePlus Real-Time PCR System (Applied Biosystems, USA) using Fast Universal PCR Master Mix (Applied Biosystems) -PCR was performed. Then, the expression of myogenic markers was quantified using TaqMan Gene Expression Assays (Applied Biosystems), and then divided into beta actin values. The markers used were beta actin (Actb, Mm00607939_s1), Myogenic differentiation 1 (Myod1, Mm00435125_m1), myogenic factor 5 (Myf5, Mm00440387_m1), myogenin (myg, Mm01332564_m1), myosin heavy chain 2 (Myh2, Mm00446194_1)

As a result, as shown in FIG. 5G, the expressions of MyoD1 and Myf5, the initial markers of progenitor cell differentiation, tended to increase by day 14 and then decrease by day 21. It tended to increase. In addition, it was confirmed that CS-AF showed high expression in all four differentiation markers compared to CON.

The above description of the present invention is for illustration only, and those of ordinary skill in the art to which the present invention pertains can understand that the present invention can be easily modified to other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Claims (8)

(a) myoblast, Preparing a bioink comprising one or more cells and collagen selected from the group consisting of cardiomyoblast, smooth muscle cell, and vascular cells;
(b) arranging the collagen and cells in one direction by applying a shear stress to the bioink of step (a);
(c) dispensing the bioink of step (b) onto a plate to form an artificial structure; And
(d) curing and arranging the artificial structure of step (c) using potassium chloride-glycine solution,
The distribution of step (c) is characterized in that consisting of a flow rate of 0.7uLs ^-1 to 0.9uLs ^-1 , the method of manufacturing an arrayed cell support.
According to claim 1,
In step (a), collagen is 3 to 9% by weight based on the total weight of bioink. Characterized in that it is included, the method of manufacturing the arranged cell support.
According to claim 1,
The number of cells in step (a) is 1 x 10 ⁶ cells/mL ^-1 to 1 x 10 ⁹ cells/mL ^-1 , characterized in that the method of manufacturing an arrayed cell support.
According to claim 1,
In the step (c), the temperature of the plate is 33 °C to 40 °C, characterized in that the method of manufacturing the arranged cell support.
delete According to claim 1,
The distribution of step (c) is characterized in that consisting of a speed of 9mms ^-1 to 11mms ^-1 , the method of manufacturing the arranged cell support.
According to claim 1,
The distribution of step (c) is characterized in that the artificial structure is made to form a lattice structure, the method of manufacturing an arranged cell support.
According to claim 1,
In the potassium chloride-glycine solution of step (d), characterized in that the potassium chloride is 50 to 200 mM and the glycine is 50 to 100 mM, the method of manufacturing an arrayed cell support.

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