KR20230073749A - Febricating method of beating-type cardiac mucle patch and beating-type cardiac mucle patch fabricated by using the same - Google Patents

Abstract

The present invention relates to a method for manufacturing a pulsatile myocardial patch that maintains pulsation outside the body and a pulsatile myocardial patch manufactured using the same.

Description

Manufacturing method of pulsatile myocardial patch and pulsatile myocardial patch manufactured using the same

The present specification relates to a pulsatile myocardial patch and a manufacturing method of the pulsatile myocardial patch manufactured using the same.

As for the severity of cardiovascular disease, the prevalence and mortality of cardiovascular disease are continuously increasing in Korea due to the westernization of lifestyle and eating habits, and it currently ranks second among the causes of death in Koreans. In the case of the United States, heart disease is emerging as a serious medical issue worldwide, with 12.4 million coronary artery disease, 7.3 million myocardial infarction, and 6.4 million angina pectoris.

Limitations of treatment for myocardial infarction and congestive heart failure include acute myocardial infarction (AMI), which is known to have the highest patient increase among cardiovascular diseases, and congestive heart failure (CHF) caused by it, which is irreversible. It is a disease caused by the loss of myocardial cells, known as cells. Despite the development of various treatments such as drug treatment and angioplasty, fundamental treatment is difficult, and heart transplantation, known as the most effective treatment, is not realistic due to limitations in donors. am. Existing methods of administering stem cells for myocardial regeneration include direct injection into the myocardium, coronary injection, and intravenous injection. In addition, recently, in order to make local delivery more efficient, a method of delivering cells using a tissue-engineered three-dimensional scaffold coated with stem cells on a biocompatible matrix or biomaterial has been used.

As such, various methods for treating heart disease are being studied, and there is a need to develop means for effective treatment.

Republic of Korea Patent Publication No. 10-2008-0030026

The present specification is intended to provide a method for manufacturing a pulsatile myocardial patch that can be used for treatment of heart disease and a pulsatile myocardial patch manufactured thereby.

An exemplary embodiment of the present invention, A) preparing an alginate medium having a pattern structure having a pitch of 50 μm to 500 μm provided on at least one surface; B) first treating the alginate medium with a first modifying solution containing EDC (N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride) and NHS (N-Hydroxysuccinimide); C) secondary treatment of the alginate medium with a second modified solution containing Tyramine Hydrochloride and HEPES (4-(2-Hydroxyethyl)-1-Piperazineethanesulfonic acid); D) forming an extracellular matrix coating layer on one side of the alginate medium; and E) seeding and culturing cardiomyocytes on the alginate medium on which the extracellular matrix coating layer is formed to form a myocardial cell layer.

Another embodiment of the present invention provides a pulsatile myocardial patch manufactured using the above manufacturing method.

Since the pulsatile myocardial patch manufactured according to the present invention maintains a pulsatile state even outside the body, it may be used for heart disease treatment or for heart disease treatment research.

Other objects and advantages of the present invention may be more clearly understood by the following detailed description, claims and drawings.

1 is a schematic diagram illustrating a manufacturing process of a pulsatile myocardial patch according to an embodiment.
Figure 2 shows an enlarged image of an alginate medium prepared according to an embodiment.
3 shows a microscopic image of a pulsatile myocardial patch prepared according to an example.

In this specification, when a member is said to be located “on” another member, this includes not only the case where a member is in contact with another member, but also the case where another member exists between the two members.

In this specification, when a part is said to "include" a certain component, it means that it may further include other components without excluding other components unless otherwise stated.

The present inventors have confirmed that when an alginate-based medium is modified step by step and cardiomyocytes are cultured, a myocardial patch that maintains beating in vitro, like myocardial cells in a human body, can be prepared, and the following inventions are provided. came to do Unlike existing scaffolds for treating heart disease, the pulsatile myocardial patch according to the present invention behaves more like myocardial cells in the body, so it is expected that it can be used to effectively treat heart disease. In addition, since the pulsatile myocardial patch according to the present invention has a unique behavior of beating even outside the body, it can be used for experimental purposes for treating heart disease outside the body.

Hereinafter, the present invention will be described in detail.

An exemplary embodiment of the present invention, A) preparing an alginate medium having a pattern structure having a pitch of 50 μm to 500 μm provided on at least one surface; B) first treating the alginate medium with a first modifying solution containing EDC (N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride) and NHS (N-Hydroxysuccinimide); C) secondary treatment of the alginate medium with a second modified solution containing Tyramine Hydrochloride and HEPES (4-(2-Hydroxyethyl)-1-Piperazineethanesulfonic acid); D) forming an extracellular matrix coating layer on one side of the alginate medium; and E) seeding and culturing cardiomyocytes on the alginate medium on which the extracellular matrix coating layer is formed to form a myocardial cell layer.

Step A): Preparing an alginate medium

According to one embodiment of the present invention, the alginate medium may be an alginate-based medium. In addition, the alginate medium may be a medium prepared using an alginate concentrate, and may include a pattern region having a pattern structure on at least one surface of the alginate medium. For example, the alginate concentrate may be dissolved in about 1 vol% of alginate in a sodium chloride solution.

According to an exemplary embodiment of the present invention, in step A), a1) using a 3D printer, an alginate structure including a first layer of a plate and a second layer including a plurality of pattern structures on the first layer Preparing a; and a2) preparing the alginate medium by curing the alginate structure using a curing agent.

According to one embodiment of the present invention, step a1) may be to form the first layer and the second layer using an ink composition containing alginate and a 3D printer. An ink composition containing alginate that is ejected using the 3D printer may have low viscosity, making it difficult to form the pattern structure of the second layer.

Therefore, according to one embodiment of the present invention, the 3D printer is provided with a cooling tray adjusted to a temperature range of -40 ℃ to -10 ℃, the alginate structure can be produced on the cooling tray. An alginate structure having a predetermined shape may be formed in the ink composition containing the alginate discharged by the cooling tray.

Furthermore, an alginate medium may be formed by curing the alginate structure formed in step a1) using a curing agent (step a2). For example, the curing agent may be barium chloride, but is not limited thereto, and various materials capable of curing the alginate structure may be used.

According to one embodiment of the present invention, when forming the second layer in step a1), the ejection speed of the alginate solution of the 3D printer is 0.01 mm / s to 0.2 mm / s, and the printing speed is 2 mm / s to 10 mm/s. When the ejection speed and the printing speed are within the above ranges, the pattern structure of the second layer may be formed while stably maintaining a predetermined pattern spacing (pitch), pattern line width, and/or pattern height. Furthermore, the pattern structure thus formed may have a pitch of 50 μm to 500 μm, 100 μm to 400 μm, 250 μm to 350 μm, or about 300 μm on the alginate medium, and when it has a pitch within the above range, the cultured Myocardial cell layer can maintain beating even outside the body.

According to an exemplary embodiment of the present invention, the width of the pattern structure of the alginate medium may be 50 μm to 500 μm, 100 μm to 400 μm, 250 μm to 350 μm, or about 300 μm. The width of the pattern structure may mean the width of the pattern structure on a top view of the alginate medium, and the width range of the pattern structure may be the same as or similar to the pitch range of the pattern structure.

Further, according to an exemplary embodiment of the present invention, the height of the pattern structure of the alginate medium may be 50 μm to 250 μm, 100 μm to 200 μm, or 100 μm to 150 μm. When the height of the pattern structure is within the above range, a myocardial cell layer by seeded myocardial cells may be continuously formed.

When a myocardial patch is prepared using an alginate medium having a pattern structure having the above-described range, beating outside the body can be maximized.

According to an exemplary embodiment of the present invention, the pattern structure may form various types of patterns, for example, a linear pattern or a mesh pattern may be formed on the alginate medium.

Step B): first treating the alginate medium with a first modified solution

According to an exemplary embodiment of the present invention, in step B), the alginate medium is treated with a first modified solution containing EDC (N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride) and NHS (N-Hydroxysuccinimide). Thus, the carboxyl group of the alginate medium may be modified with a primary amine group. Specifically, the step B) may be to activate the carboxyl group bound to the alginate molecule of the alginate medium through an EDC coupling reaction through an EDC-NHS activation process so that it binds to NHS containing an amine group. Through this, the primary treated alginate medium can be converted into a sulfo-NHS ester through a secondary treatment described below. That is, when the secondary treatment is performed without the primary treatment, a problem in that the protein adsorption activation effect according to the secondary treatment is not sufficiently expressed may occur.

According to an exemplary embodiment of the present invention, in the first modified solution, the weight ratio of the NHS to the EDC may be 5:1 to 10:1. Specifically, the weight ratio of the NHS to the EDC may be 6:1 to 8:1, or 6:1 to 7:1. In addition, in the first modified solution, the concentration of the EDC may be 10 mM to 100 mM, and the concentration of the NHS may be 100 mM to 500 mM. Within the above weight ratio range and/or within the above concentration range, the carboxyl groups of the alginate medium can be effectively converted into primary amine groups. Specifically, when treated within the above weight ratio range and/or within the above concentration range, tyramine may be smoothly attached in the second treatment of the alginate medium with the second modified solution.

Step C): Secondary treatment of the alginate medium with a second modified solution

According to an exemplary embodiment of the present invention, in step C), the alginate medium is treated with a second modified solution containing tyramine hydrochloride and HEPES (4-(2-Hydroxyethyl)-1-Piperazineethanesulfonic acid). Thus, the amine group of the alginate medium modified through the first treatment and the tyramine of the tyramine hydrochloride may be amidated so that they covalently bond with each other. Tyramine bound to the alginate medium can improve the binding with tyrosine, one of the components of protein, and increase the content of the protein bound to the surface of the alginate medium. As the amount of protein bound to the surface of the alginate medium increases, cell adhesion may be facilitated on the surface of the alginate medium. That is, when the first treatment and the second treatment are sequentially performed, the alginate medium effectively adsorbs proteins, so that protein-based materials such as myocardial cells and extracellular matrix can be adhered and cultured smoothly.

According to an exemplary embodiment of the present invention, in the second modified solution, the molar ratio of the tyramine hydrochloride and the HEPES may be 1:2 to 2:1. Specifically, the molar ratio of the tyramine hydrochloride and the HEPES may be 1.5:1 to 1:1.5, or 1:1. Furthermore, in the second modified solution, the concentration of tyramine hydrochloride may be 0.01 g/ml to 0.5 g/ml, and the concentration of HEPES may be 0.05 M to 0.25 M. At the molar ratio of the tyramine hydrochloride and the HEPES and/or the concentration of the tyramine hydrochloride and the HEPES within the above range, the alginate medium can effectively activate protein adhesion. That is, the adhesiveness of the extracellular matrix material increases within the above molar ratio range and/or concentration range, so that the extracellular matrix coating layer can be prevented from falling off after formation.

According to an exemplary embodiment of the present invention, the second modified solution may further include a pH adjusting agent. Specifically, the pH adjusting agent maintains the pH of the second modified solution at a neutral level of 7.0 to 7.5, specifically 7.2 to 7.4, so that the extracellular matrix material such as Matrigel facilitates attachment on the alginate medium can do.

Step D): Forming an extracellular matrix coating layer on one side of the alginate medium

According to an exemplary embodiment of the present invention, step D) may be to form an extracellular matrix coating layer on at least one side of the alginate medium on which the pattern structure is formed in the alginate medium subjected to the first and second treatments.

The extracellular matrix coating layer may be formed using an extracellular matrix generally available in the art. For example, an extracellular matrix solution is applied on the alginate medium, or the alginate medium is applied to the extracellular matrix solution. It can be formed by impregnating. For example, the extracellular matrix may use Matrigel®, but is not limited thereto, and various types of extracellular matrix may be used.

The extracellular matrix layer may serve to improve adhesion and mass transfer between the myocardial cell layer derived from myocardial cells seeded on its surface and the alginate medium.

Step E): Forming the myocardial cell layer

According to an exemplary embodiment of the present invention, step D) may include seeding myocardial cells on the extracellular matrix coating layer to form a myocardial cell layer. Specifically, cardiomyocytes may be seeded on the extracellular matrix coating layer on one surface of the alginate medium having the pattern structures, and more specifically, the cardiomyocytes may be seeded between the pattern structures of the alginate medium.

According to one embodiment of the present invention, the cardiomyocytes may be derived from induced pluripotent stem cells (iPSCs). The method of inducing cardiomyocytes from iPSCs may be performed using a variety of known methods.

The seeded myocardial cells may secrete an extracellular matrix through a culture process to form a myocardial cell layer. The myocardial cell layer thus formed may exist while beating on the alginate medium. Specifically, according to an exemplary embodiment of the present invention, the pulsatile myocardial patch may beat 50 times/minute to 100 times/minute.

An exemplary embodiment of the present invention provides a pulsatile myocardial patch manufactured using the above manufacturing method. Unlike conventional materials using cardiomyocytes for treating heart disease, the pulsatile myocardial patch maintains its heartbeat even outside the body, and thus is expected to effectively treat heart disease. In addition, after the pulsatile myocardial patch according to the present invention is manufactured according to the patient's needs, it can be used for the treatment of heart disease by replacing or supplementing the heart tissue in a local area. Furthermore, the pulsatile myocardial patch according to the present invention may be used for research purposes for heart treatment and help in the development of methods for treating heart disease.

Hereinafter, examples will be described in detail to explain the present invention in detail. However, embodiments according to the present invention can be modified in many different forms, and the scope of the present invention is not construed as being limited to the embodiments described below. The embodiments herein are provided to more completely explain the present invention to those skilled in the art.

[Example]

1 is a schematic diagram illustrating a manufacturing process of a pulsatile myocardial patch according to an embodiment. Specifically, FIG. 1 illustrates the following processes of preparation of an alginate medium, modification of the alginate medium, formation of an extracellular matrix coating layer, and seeding of cardiomyocytes, and formation of a myocardial cell layer after seeding of cardiomyocytes is not shown.

(1) Preparation of alginate medium

After storing 1% alginate (LT12/LN18, Alginatec GmbH, Riedenheim, Germany) stored at 4 ° C. for 1 hour at room temperature (RT), a 3D printer equipped with a cooling tray maintaining about -20 ° C. INVIVO, ROKIT Healthcare) was transferred to the syringe module. The nozzle size of the 3D printer was 23 gauge, the syringe ejection speed of the 3D printer was set to 0.05 mm/s, and the printing speed (moving speed of the bed plate) was set to 5 mm/s on the cooling tray. An alginate structure was formed. Then, a barium chloride solution was applied on the alginate structure using a pipette and cured at RT for about 30 minutes, after which residual barium chloride was removed with a sodium chloride solution. The diameter of the alginate medium prepared through this process was about 15 mm, and the pattern spacing was about 300 μm.

Figure 2 shows an enlarged image of an alginate medium prepared according to an embodiment. The dark line in FIG. 2 means a boundary where the pattern structures are formed, and it can be confirmed that the pattern structures are uniformly formed with the interval between the pattern structures being about 300 μm and the width of the pattern structures being about 300 μm.

(2) Modification of alginate medium and formation of extracellular matrix coating layer

After dissolving 1.9 g of NHS (50 mM, Sigma Aldrich) and 0.28 g of EDC (200 mM, Sigma Aldrich) in 50 ml of distilled water, the prepared alginate medium was placed in a negative pressure filtered solution at RT for about 30 minutes. Impregnated in , the first modification was performed. Furthermore, secondary modification was performed by impregnating the primary modified alginate medium with a solution in which Tyramine hydrochloride and HEPES were dissolved in distilled water at a molar ratio of about 1:1 for about one day at RT. Then, after removing the residue with sodium chloride solution, the primary and secondary modified alginate medium was mixed with 5 mg of Matrigel (corning, Basement Membrane Matrix High Concentration) in 60 ml of DMEM/F-12 (HEPES, Gibco) The mixed solution was impregnated at RT for about 1 hour to form an extracellular matrix coating layer.

(3) Formation of myocardial cell layer

In the space between the pattern structures on the extracellular matrix coating layer, iPSC-derived cardiomyocytes (1 × 10 ⁶ cells) were seeded, and in an incubator with a humidity of about 95 RH%, about 37 ° C, and about 5% CO ₂ atmosphere. by culturing in a myocardial cell layer to form a pulsatile myocardial patch.

3 shows a microscopic image of a pulsatile myocardial patch prepared according to an example. The pulsatile cardiomyocytes prepared as described above existed while maintaining beats of 50 times/min to about 100 times/min.

Claims (12)

A) preparing an alginate medium provided on at least one surface with a pattern structure having a pitch of 50 μm to 500 μm;
B) first treating the alginate medium with a first modifying solution containing EDC (N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride) and NHS (N-Hydroxysuccinimide);
C) secondary treatment of the alginate medium with a second modified solution containing Tyramine Hydrochloride and HEPES (4-(2-Hydroxyethyl)-1-Piperazineethanesulfonic acid);
D) forming an extracellular matrix coating layer on one side of the alginate medium; and
E) seeding and then culturing cardiomyocytes on the alginate medium on which the extracellular matrix coating layer is formed to form a myocardial cell layer;
Manufacturing method of pulsatile myocardial patch. The method according to claim 1, wherein the step A),
a1) using a 3D printer, manufacturing an alginate structure including a plate-shaped first layer and a second layer including a plurality of pattern structures on the first layer; and
a2) preparing the alginate medium by curing the alginate structure using a curing agent. The method of claim 2,
The method of manufacturing a pulsatile myocardial patch, wherein the 3D printer has a cooling tray controlled in a temperature range of -40 ° C to -10 ° C, and the alginate structure is manufactured on the cooling tray. The method of claim 2,
When forming the second layer in step a1), the discharge speed of the alginate solution of the 3D printer is 0.01 mm / s to 0.2 mm / s, and the printing speed is 2 mm / s to 10 mm / s , Manufacturing method of pulsatile myocardial patch. The method of claim 1,
The method of manufacturing a pulsatile myocardial patch, wherein the pattern structure of the alginate medium has a width of 50 μm to 500 μm. The method of claim 1,
The method of manufacturing a pulsatile myocardial patch, wherein the height of the pattern structure of the alginate medium is 50 μm to 250 μm. The method of claim 1,
In the first modified solution, the weight ratio of the NHS to the EDC is 5: 1 to 10: 1, the manufacturing method of the pulsatile myocardial patch. The method of claim 1,
In the second modified solution, the molar ratio of the tyramine hydrochloride and the HEPES is 1:2 to 2:1, the manufacturing method of the pulsatile myocardial patch. The method of claim 1,
The method of manufacturing a pulsatile myocardial patch, wherein the cardiomyocytes are derived from induced pluripotent stem cells (iPSCs). The method of claim 1,
The method of manufacturing a pulsatile myocardial patch, wherein the cardiomyocytes in step E) are seeded between the patterned structures of the alginate medium. The method of claim 1,
The method of manufacturing a pulsatile myocardial patch, wherein the pulsatile myocardial patch beats at 50 times/min to 100 times/min. A pulsatile myocardial patch manufactured using the manufacturing method according to claim 1.

Images