Hyaluronic acid-based cell spheroid containing iron oxide and manufacturing method therefor

Abstract

The present invention relates to a hyaluronic acid-based cell spheroid containing iron oxide and a manufacturing method therefor. By introducing hyaluronic acid particles containing magnetic iron oxide and inducing physical stimulation thereto, vascularization and osteogenic differentiation ability of the cell spheroid can be improved.

Description

Hyaluronic acid-based cell spheroids containing iron oxide and methods for manufacturing the same

[0001] The present invention relates to a hyaluronic acid-based cell spheroid containing iron oxide and a method for manufacturing the same.

[0002] Bone tissue is a highly vascularized tissue within the body, and adequate blood flow is one of the essential factors to consider during the bone regeneration process. The vascular network within bone tissue supplies oxygen and nutrients and facilitates the removal of waste products. Therefore, smooth blood flow plays a crucial role in supporting the formation and maturation of new bone tissue during the bone regeneration process.

[0003] However, the understanding of the composition of bone vascular connections and growth-mediating processes is not yet clear. The specific mechanisms regarding which cells exchange signals and how they interact during the formation and regeneration of bone blood vessels have not yet been elucidated. This acts as a major obstacle in developing effective methods to promote angiogenesis and regeneration.

[0004] Against this backdrop, research mimicking the vascularization environment of bone faces many challenges. It is extremely difficult to reproduce the complex processes of blood vessel formation and maintenance in vivo within a laboratory setting. To mimic vascularized bone tissue, it is necessary to implement complex three-dimensional structures containing various cell types and biochemical signals, while maintaining conditions identical to those in vivo. With current technology, it is very difficult to perfectly mimic the vascularization environment of bone tissue.

[0005] Research on spheroids, three-dimensional cell aggregates that more closely mimic the in vivo microenvironment, occupies a very important position in the fields of modern life science and regenerative medicine. Spheroids provide an environment where cells can grow and interact freely, closer to their natural state. This helps maintain higher cellular functions than in monolayer culture. In particular, spheroids can better preserve the physiological characteristics of cells by providing a structure similar to the extracellular matrix that cells experience in their natural environment. This helps overcome limitations observed in monolayer culture, such as the loss of self-renewal and low differentiation capacity. These spheroids can contribute to reproducing intercellular interactions, interactions between cells and the extracellular matrix, and complex intercellular signaling networks that can significantly influence the functional maturation and differentiation of cells. (Tissue Engineering and Regenerative Medicine., 2024, 1-12).

[0006] Therefore, co-culture of endothelial and mesenchymal cells is currently widely used to produce vascularized bone spheroids. However, this co-culture method faces various limitations and challenges. The most significant problem is that the vascular structure cannot be maintained within the spheroid for an extended period. A spheroid is a structure in which cells are assembled in three dimensions to form a spherical shape; if sufficient blood vessels are not formed within it, the cells cannot receive adequate nutrients and oxygen. This leads to oxygen deficiency in the center of the spheroid, which can ultimately lower cell viability and reduce the effectiveness of the study.

[0007] In addition, oxygen deficiency caused by the dense concentration of cells within the spheroid is another significant problem. In a densely packed environment, the diffusion of oxygen and nutrients is restricted, leading to hypoxia and nutrient deficiency in the center of the spheroid. This environment not only hinders cell growth and function but also makes it difficult to obtain desired experimental results. In particular, this oxygen deficiency becomes a major obstacle when attempting to accurately maintain cellular physiological conditions.

[0008] Therefore, various approaches are being studied to address these problems. For example, methods such as forming artificial vascular structures within spheroids or developing new culture techniques to improve the supply of oxygen and nutrients are being attempted. Additionally, research is being conducted to control cell density or optimize spheroid size. These studies are expected to play an important role in increasing the efficiency of co-culture between cells and mesenchymal stem cells and in obtaining more stable and reproducible results.

[0009] [Prior Art Literature]

[0010] [Patent Literature]

[0011] KR 10-2023-0164993 A

[0012] The object of the present invention is to provide a hyaluronic acid-based cell spheroid containing iron oxide and a method for manufacturing the same.

[0013] Another objective of the present invention is to provide cell spheroids with improved angiogenesis and osteodifferentiation capabilities by introducing hyaluronic acid particles containing magnetic iron oxide and inducing physical stimulation thereto.

[0014] To achieve the above objective, a hyaluronic acid-based cell spheroid containing iron oxide according to one embodiment of the present invention comprises hyaluronic acid particles containing iron oxide and cells.

[0015] The above cells are selected from a group consisting of endothelial cells, mesenchymal stem cells, and mixtures thereof.

[0016] The above cell spheroids are excellent in bone differentiation and vascularization ability when physical stimulation is induced.

[0017] The aspect ratio of the above cell spheroid is 1.0 to 1.2.

[0018] A method for preparing cell spheroids based on hyaluronic acid containing iron oxide according to another embodiment of the present invention comprises the steps of: preparing hyaluronic acid particles containing iron oxide; mixing and culturing the hyaluronic acid particles containing iron oxide in a cell suspension to form spheroids in a culture; and stirring the culture to induce physical stimulation.

[0019] The step of inducing the above physical stimulation is to improve the osteogenic differentiation and vascularization capabilities of the cell spheroid.

[0020] The step of preparing hyaluronic acid particles containing iron oxide comprises: mixing hyaluronic acid, iron oxide, and a crosslinking agent in a basic aqueous solution to obtain a polymer aqueous solution; mixing the polymer aqueous solution in an oil phase in which a surfactant is dissolved to form a w / o emulsion; stirring the w / o emulsion to crosslink the hyaluronic acid and iron oxide; neutralizing the w / o emulsion after the stirring step; and separating the hyaluronic acid particles containing iron oxide from the w / o emulsion.

[0021] The crosslinking agent is selected from the group consisting of polyethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, butylene glycol diglycidyl ether, ethylene glycol diglycidyl ether, 1,6-hexanediol diglycidyl ether, propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, polytetramethylglycol diglycidyl ether, neopentyl glycol diglycidyl ether, polyglycerol polyglycidyl ether, diglycerol polyglycidyl ether, glycerol polyglycidyl ether, trimethylpropane polyglycidyl ether, 1,2-(bis(2,3-epoxypropoxy)ethylene), pentaerythritol polyglycidyl ether, sorbitol polyglycidyl ether, and mixtures thereof.

[0022] The above surfactant is selected from the group consisting of cetyl PEG / PPG-10 / 1 dimethicone (ABIL EM-90), sorbitan sesquioleate (ARLACEL 83), polyethylene glycol (30) dipolyhydroxy stearate (ARLACEL P135)) and mixtures thereof.

[0023] The above oil phase is selected from the group consisting of cetyl ethylhexanoate, dodecane, heptane, and mixtures thereof.

[0024] A bone tissue regeneration composition according to another embodiment of the present invention comprises a hyaluronic acid-based cell spheroid containing iron oxide.

[0025] The present invention relates to a hyaluronic acid-based cell spheroid containing iron oxide and a method for manufacturing the same. By introducing hyaluronic acid particles containing magnetic iron oxide and inducing physical stimulation thereto, the angiogenesis and osteogenic differentiation ability of the cell spheroid can be improved.

[0026] FIG. 1 is an overall schematic diagram of a method for preparing cell spheroids containing hyaluronic acid containing iron oxide using physical stimulation according to one embodiment of the present invention.

[0027] FIG. 2 is a schematic diagram and results of a process for producing hyaluronic acid particles containing iron oxide according to one embodiment of the present invention.

[0028] Figure 3 is the result of confirming the shape and size of a cell spheroid containing hyaluronic acid particles containing iron oxide according to one embodiment of the present invention.

[0029] Figure 4 is the result of confirming the survival rate of cell spheroids containing hyaluronic acid particles containing iron oxide according to one embodiment of the present invention.

[0030] Figure 5 is the result of confirming the enhanced angiogenesis ability of cell spheroids containing iron oxide-containing hyaluronic acid using physical stimulation according to one embodiment of the present invention through mRNA expression levels via immunofluorescence staining and RT-PCR.

[0031] Figure 6 is the result of confirming the enhanced osteogenic differentiation ability of cell spheroids containing iron oxide-containing hyaluronic acid using physical stimulation according to one embodiment of the present invention by immunofluorescence staining and RT-PCR.

[0032] The present invention relates to hyaluronic acid particles containing iron oxide and hyaluronic acid-based cell spheroids containing iron oxide containing cells.

[0033] Hereinafter, embodiments of the present invention are described in detail so that those skilled in the art can easily implement the invention. However, the present invention may be embodied in various different forms and is not limited to the embodiments described herein.

[0034] In this specification, the term "room temperature" means a temperature range condition of 20 to 30°C.

[0035] Meanwhile, in this specification, the term "MSC / HUVEC" refers to mesenchymal stem cells (MSC) and human umbilical vein endothelial cells (HUVEC), and "MSC / HUVEC spheroid" refers to a spheroid formed through the co-culture of said mesenchymal stem cells and human umbilical vein endothelial cells.

[0036] Bone spheroids, which are used in applications of regenerative medicine and tissue engineering, are primarily produced through the co-culture of endothelial cells and mesenchymal cells as mentioned above. However, in this case, there are issues such as the inability to maintain vascular structures within the spheroid for a long time or oxygen deficiency due to cell density, so various studies are being conducted to solve these problems, such as optimizing culture techniques.

[0037] The present invention provides a bone spheroid and a method for manufacturing the same, which can exhibit improved vascularization and bone differentiation capabilities compared to existing MSC / HUVEC spheroids by using iron oxide particles having magnetism capable of inducing physical stimulation and inducing physical stimulation of the iron oxide during the manufacturing process of the spheroid, and intends to apply the same to regenerative medicine and tissue engineering.

[0038] A hyaluronic acid-based cell spheroid containing iron oxide according to one embodiment of the present invention comprises hyaluronic acid particles containing iron oxide and cells.

[0039] The above cells are selected from a group consisting of endothelial cells, mesenchymal stem cells, and mixtures thereof.

[0040] Specifically, the endothelial cells are cells that constitute the inner wall of blood vessels, form a boundary between blood and blood vessel structures, and perform various physiological and pathological functions; they may be endothelial cells selected from the group consisting of Human Umbilical Vein Endothelial Cells (HUVEC), Human Microvascular Endothelial Cells (HMEC-1), Human Coronary Artery Endothelial Cells (HCAEC), and Human Aortic Endothelial Cells (HAEC). However, they are not limited to the above examples and are defined to include all endothelial cells capable of forming a spheroid structure to mimic intercellular interactions, angiogenesis, and various physiological environments. More specifically, the endothelial cells are Human Umbilical Vein Endothelial Cells (HUVEC).

[0041] Meanwhile, the above-mentioned mesenchymal stem cells (MSCs) refer to cells found in various tissues and organs that have the ability to differentiate into various cells. Specifically, the above-mentioned mesenchymal stem cells may be selected from the group consisting of bone marrow-derived stem cells (BMSCs), adipose-derived stem cells (ADSCs), umbilical cord stem cells, placental stem cells, liver-derived stem cells, cardiac stem cells, and neural stem cells. However, the above examples are not limited, and the mesenchymal stem cells are not particularly restricted as long as they are mesenchymal stem cells that a person skilled in the art can co-culture with endothelial cells to form spheroids.

[0042] Preferably, the cells are mesenchymal stem cells and endothelial cells. More specifically, they are osteogenic mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs), which can be defined as "MSC / HUVEC".

[0043] When MSC / HUVEC is used as the above cell, it is possible to form bone spheroids using mesenchymal stem cells capable of bone differentiation.

[0044] Preferably, the hyaluronic acid particles containing iron oxide are included in an amount of 10 to 30 weight percent relative to the cells. Within this range, physical stimulation can be effectively induced to maximize the osteogenic differentiation and angiogenic abilities of the spheroids, whereas outside this range, the interaction between cells weakens, resulting in the spheroids not clumping together or a decrease in osteogenic differentiation and angiogenic abilities.

[0045] Specifically, if the above range is exceeded, the content of hyaluronic acid particles becomes relatively higher than the cell content, and the interaction between cells weakens, so spheroids may not be formed. On the other hand, if the above range is below, some spheroids are formed, but the content of hyaluronic acid particles is too low, so the formation of uniform spheroids may be limited, and the bone differentiation ability and vascularization ability may be reduced.

[0046] The above cell spheroids are excellent in bone differentiation and vascularization ability when physical stimulation is induced.

[0047] The above cell spheroid is characterized by containing hyaluronic acid particles containing iron oxide. Since the iron oxide exhibits supermagnetic properties, when physical stimulation is induced therein, it can induce spin in the spheroid and facilitate the supply of nutrients and oxygen into the spheroid, and ultimately improve bone differentiation ability and vascularization ability.

[0048] The aspect ratio of the above cell spheroid is 1.0 to 1.2.

[0049] Specifically, the aspect ratio of the cell spheroid is 1.0 to 1.15, more specifically 1.0 to 1.12, and even more specifically 1.0 to 1.06. When the cell spheroid is formed within the above aspect ratio range, it can exhibit superior osteodifferentiation and angiogenesis capabilities.

[0050] A method for preparing a cell spheroid containing hyaluronic acid containing iron oxide according to one embodiment of the present invention comprises the steps of: preparing hyaluronic acid particles containing iron oxide; mixing and culturing the hyaluronic acid particles containing iron oxide in a cell suspension to form a spheroid in a culture; and stirring the culture to induce physical stimulation.

[0051] The above "culture" can be carried out by methods known in the art depending on the type of cell, and preferably means culturing in an incubator that provides a growth environment of 30 to 37°C, 1-10% O2, and 5% CO2.

[0052] The hyaluronic acid particles described above have the characteristic of forming spaces between cells within the spheroid to alleviate the problem of oxygen deficiency and allow cells to survive and grow for a longer period. Furthermore, the hyaluronic acid particles bind to iron oxide to increase the biocompatibility of iron oxide, thereby enabling iron oxide to be safely utilized within the spheroid.

[0053] The molecular weight of the hyaluronic acid is 300,000 to 10,000,000 based on the number average molecular weight, and preferably 700,000 to 1,500,000.

[0054] Meanwhile, the iron oxide mentioned above has supermagnetic properties, and when physical stimulation is induced through stirring on a spheroid containing hyaluronic acid particles containing it, it has the characteristic of promoting nutrient supply inside the spheroid forming the spheroid and ultimately improving bone differentiation ability and vascularization ability.

[0055] Preferably, the hyaluronic acid particles containing iron oxide can be mixed in a cell suspension at a weight percentage of 10 to 30% to form spheroids. Within this range, physical stimulation can be effectively induced to maximize the osteogenic differentiation and angiogenic capabilities of the spheroids, whereas outside this range, the interaction between cells weakens, and they may not clump together into spheroids.

[0056] The step of inducing physical stimulation in the above culture corresponds to a stirring step for improving osteodifferentiation ability and vascularization ability by applying physical stimulation to spheroids containing iron oxide within the culture, thereby inducing mechanical stress and spin in the spheroids, supplying oxygen and nutrients into the spheroids, and stimulating vascular factors by promoting cell interaction.

[0057] Preferably, the physical stimulation means stirring the culture at a speed of 50 to 100 rpm for 20 to 60 minutes daily at a temperature of 20 to 40°C, and more specifically, means stirring the culture at a speed of 80 rpm for 30 minutes at the same time for 2 to 14 days at a temperature of 30°C.

[0058] Within the above range, when physical stimulation is applied, the spin-inducing effect on the spheroid and the effect of supplying oxygen and nutrients into the spheroid can be enhanced, thereby further improving osteogenicity and vascularization ability.

[0059] On the other hand, if the above physical stimulation is applied for more than 1 hour a day, it may have an adverse effect on the survival rate of the spheroid due to the problem of not receiving an incubator environment for a long period and the prolonged stimulation.

[0060] More preferably, the physical stimulation may be performed twice a day at 6-hour intervals while maintaining the stirring conditions, thereby further maximizing the osteodifferentiation and vascularization capabilities intended in the present invention. The step of inducing the physical stimulation is to improve the osteodifferentiation and vascularization capabilities of the cell spheroids.

[0061] The present invention is characterized by introducing iron oxide nanoparticles having supermagnetic properties capable of inducing physical stimulation into hyaluronic acid particles as described above, and by inducing physical stimulation through stirring during the spheroid manufacturing process, thereby improving the osteogenic differentiation and angiogenic ability of the cell spheroids formed therein.

[0062] More specifically, the present invention is characterized by the ability to induce spin by introducing iron oxide nanoparticles with hyaline properties and inducing physical stimulation, unlike spheroids formed by the co-culture of existing endothelial cells and mesenchymal stem cells. Through this, not only can the interaction between endothelial cells and mesenchymal stem cells be enhanced, but nutrient supply to the spheroids forming the spheroids can also be promoted, thereby enabling the formation of bone spheroids with improved bone differentiation and vascularization capabilities compared to existing spheroid models.

[0063] The step of preparing hyaluronic acid particles containing iron oxide comprises: mixing hyaluronic acid, iron oxide, and a crosslinking agent in a basic aqueous solution to obtain a polymer aqueous solution; mixing the polymer aqueous solution in an oil phase in which a surfactant is dissolved to form a w / o emulsion; stirring the w / o emulsion to crosslink the hyaluronic acid and iron oxide; neutralizing the w / o emulsion after the stirring step; and separating the hyaluronic acid particles containing iron oxide from the w / o emulsion.

[0064] The step of preparing hyaluronic acid particles containing iron oxide according to the present invention is specifically illustrated in FIG. 2.

[0065] Referring to FIG. 2, in order to manufacture hyaluronic acid particles containing iron oxide, a polymer aqueous solution is first prepared by mixing hyaluronic acid, iron oxide, and a crosslinking agent in a basic aqueous solution. Meanwhile, in order for the crosslinking reaction of the hyaluronic acid and iron oxide to proceed in the aqueous solution through the crosslinking agent, it is necessary to raise the pH of the basic aqueous solution in which the hyaluronic acid and the crosslinking agent are dissolved to 12 to 14 using a base such as sodium hydroxide, potassium hydroxide, sodium bicarbonate, or ammonia to increase the reactivity of the hydroxyl groups of the hyaluronic acid. However, since the likelihood of hydrolysis increases if the hyaluronic acid is left in such a basic aqueous solution for a long time, it is desirable to ensure that the hyaluronic acid, iron oxide, and the crosslinking agent are completely dissolved as quickly as possible when dissolving them in the basic aqueous solution.

[0066] Preferably, the content of the hyaluronic acid is 1 to 10 weight% with respect to the basic aqueous solution, and the content of the crosslinking agent is 0.01 to 1 weight% with respect to the basic aqueous solution. Meanwhile, iron oxide is included in an amount of 1 to 15 weight% of the total weight of the hyaluronic acid and the crosslinking agent.

[0067] Subsequently, the above-mentioned polymer aqueous solution is mixed onto an oil in which a surfactant is dissolved and homogenized to form a w / o emulsion, and then the w / o emulsion is stirred at 50 to 60°C to cross-link hyaluronic acid and iron oxide.

[0068] Preferably, the surfactant is included in an amount of 1 to 10 weight percent based on the total weight of the mixture of the oil phase and the aqueous phase of the w / o emulsion. The concentration of the surfactant affects the size and stability of the w / o emulsion. If the amount exceeds the above range, the w / o emulsion particles can be stabilized, but there is a problem with a large amount of residual particulate impurities in the final product. If the amount is below the above range, the particle size increases and the stability of the particles may also become unstable.

[0069] Meanwhile, following the stirring step for the cross-linking described above, a step of neutralizing the w / o emulsion that has undergone the stirring step is carried out. Specifically, the neutralization step is intended to minimize the aforementioned hydrolysis problem and refers to a step of adjusting the reaction temperature to room temperature and adding and maintaining an acid such as acetic acid, hydrochloric acid, sulfuric acid, nitric acid, or citric acid to neutralize the aqueous solution in a basic state.

[0070] Subsequently, a step can be performed to remove impurities such as oil, surfactants, and unreacted substances from the w / o emulsion that has undergone a neutralization process, and to separate only the hyaluronic acid particles containing iron oxide.

[0071] To specify the step of isolating only the hyaluronic acid particles containing iron oxide, the w / o emulsion that has undergone a neutralization process is precipitated in acetone. Then, impurities such as oil, surfactants, and unreacted substances are removed using a co-solvent of acetone and water, followed by a washing step with acetone. Subsequently, a vacuum filtration step is performed, and by vacuum drying, the hyaluronic acid particles containing iron oxide, from which residual solvent has been completely removed, can be isolated.

[0072] Preferably, the method may further include the step of swelling the separated hyaluronic acid particles containing iron oxide in distilled water and then sieving them.

[0073] Through this, iron oxide-containing hyaluronic acid particles of a uniform size can be obtained, thereby improving the efficiency of spheroid formation. Meanwhile, the particles filtered through the sieve can be washed, filtered under reduced pressure, and then vacuum dried to completely remove residual solvent.

[0074] The crosslinking agent is selected from the group consisting of polyethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, butylene glycol diglycidyl ether, ethylene glycol diglycidyl ether, 1,6-hexanediol diglycidyl ether, propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, polytetramethylglycol diglycidyl ether, neopentyl glycol diglycidyl ether, polyglycerol polyglycidyl ether, diglycerol polyglycidyl ether, glycerol polyglycidyl ether, trimethylpropane polyglycidyl ether, 1,2-(bis(2,3-epoxypropoxy)ethylene), pentaerythritol polyglycidyl ether, sorbitol polyglycidyl ether, and mixtures thereof.

[0075] The above crosslinking agent is included to form a crosslink between hyaluronic acid and iron oxide, but is not limited to the above examples, and is not particularly limited to an epoxide crosslinking agent having two or more epoxy reactive groups to crosslink hyaluronic acid and iron oxide.

[0076] The above surfactant is selected from the group consisting of cetyl PEG / PPG-10 / 1 dimethicone (ABIL EM-90), sorbitan sesquioleate (ARLACEL 83), polyethylene glycol (30) dipolyhydroxy stearate (ARLACEL P135)) and mixtures thereof.

[0077] However, the types of surfactants are not limited to the examples above, and are defined to include all surfactants that can stabilize the w / o emulsion and that a person skilled in the art can apply to the manufacturing process of the spheroid.

[0078] The above oil phase is selected from the group consisting of cetyl ethylhexanoate, dodecane, heptane, and mixtures thereof.

[0079] A bone tissue regeneration composition according to another embodiment of the present invention comprises cell spheroids containing hyaluronic acid containing iron oxide.

[0080] However, the application fields of cell spheroids produced by the manufacturing method of the present invention are not limited to this, and can be applied to various fields of regenerative medicine and tissue engineering.

[0081] Preparation Example

[0082] Preparation of iron oxide-containing hyaluronic acid-based cell spheroids using physical stimulation (Example 1)

[0083] 1) Preparation of hyaluronic acid particles containing iron oxide

[0084] Hyaluronic acid (molecular weight 1,000,000) was dissolved in a 0.1 N sodium hydroxide aqueous solution, and polyethylene glycol diglycidyl ether and iron oxide, which are crosslinking agents, were added and mixed thoroughly to prepare a polymer aqueous solution. Meanwhile, polyethylene glycol (30) dipolyhydroxy stearate (ARLACEL P135), a surfactant, was added to heptane in another reactor and melted at 60°C. The above aqueous polymer solution was slowly transferred to the heptane containing the dissolved surfactant, and a w / o emulsion was prepared by stirring with a homogenizer at 3,400 rpm for 20 minutes while maintaining the temperature at 60°C. Subsequently, a crosslinking reaction was carried out at 60°C for 2 hours using a mechanical stirrer. The temperature was adjusted to room temperature (20 to 25°C), acetic acid was added to neutralize the w / o emulsion, and the reaction was carried out at room temperature for 2 days.

[0085] Subsequently, the w / o emulsion was precipitated in acetone to obtain the reaction-completed iron oxide-containing hyaluronic acid particles. Then, impurities such as oil, surfactants, and unreacted materials were completely removed using a co-solvent of acetone and water, and finally, the particles were washed with acetone to remove water. Afterward, the residual solvent was completely removed by vacuum drying the iron oxide-containing hyaluronic acid particles obtained through vacuum filtration for 24 hours. Additionally, to obtain iron oxide-containing hyaluronic acid particles of a uniform size, the dried particles were swollen in triple-distilled water for 2 hours and then filtered through sieves with mesh sizes of 15, 100, and 200 μm. The filtered particles were washed with acetone, and the residual solvent was completely removed by vacuum drying the iron oxide-containing hyaluronic acid particles obtained after vacuum filtration for 24 hours. Meanwhile, Figure 2 shows a schematic diagram of the process for manufacturing iron oxide-containing hyaluronic acid particles.

[0086] 2) Preparation of cell spheroids containing the above iron oxide-containing hyaluronic acid particles

[0087] To mimic vascularized bone, iron oxide-containing hyaluronic acid-based MSC / HUVEC spheroids were cultured using human umbilical vein endothelial cells (HUVECs) and mesenchymal stem cells capable of osteodifferentiation.

[0088] Specifically, iron oxide-containing hyaluronic acid-based MSC / HUVEC spheroids were prepared using LabSparrow (Lab2Lab) with MSCs and HUVECs in a 1:1 ratio, at 0.525 x 10⁻⁶ each. 5 The cells were inoculated at a density of cells / dish, and it was confirmed that a single cell was included in the culture immediately after inoculation. Meanwhile, the hyaluronic acid particles containing iron oxide prepared above were dispensed into the MSC / HUVEC suspension at 30% of the cell volume, and the cells were cultured in an incubator under conditions of 5% CO2 and 37°C. The culture medium used was a 1:1 suspension of osteogenic differentiation medium and EGM2 medium, and osteogenic differentiation was induced by replacing the culture medium every 3 days. The spheroids aggregated 24 hours after inoculation. Subsequently, to induce physical stimulation, the culture containing the spheroids was stirred daily for 30 minutes at 30°C and 80 rpm. Meanwhile, Figure 1 is an overall schematic diagram of the process for manufacturing iron oxide-containing hyaluronic acid-based MSC / HUVEC spheroids (HA@MPS) using physical stimulation according to one embodiment of the present invention.

[0089] Preparation of iron oxide-containing hyaluronic acid-based cell spheroids using physical stimulation (Example 2)

[0090] The iron oxide-containing hyaluronic acid-based cell spheroids (HA@MPS_10) of Example 2 were prepared using the same method as in Example 2), except that the iron oxide-containing hyaluronic acid particles in Example 2) were dispensed into the MSC / HUVEC suspension at 10% of the cells.

[0091] Preparation of cell spheroids containing hyaluronic acid particles (Comparative Example 1)

[0092] Cell spheroids (HA; control) containing hyaluronic acid particles were prepared using the same method as in the above example, except that hyaluronic acid particles not containing iron oxide were used and a stirring step for physical stimulation was not performed during the spheroid preparation process.

[0093] Preparation of iron oxide-containing hyaluronic acid-based cell spheroids (Comparative Example 2)

[0094] Iron oxide-containing hyaluronic acid-based cell spheroids (HA@MP) were prepared using the same method as in the above example, except that a stirring step for physical stimulation was not performed during the spheroid preparation process.

[0095] Preparation of cell spheroids with different mixing ratios of hyaluronic acid particles containing iron oxide (Comparative Examples 3 and 4)

[0096] The iron oxide-containing hyaluronic acid-based cell spheroids (HA@MPS_50) of Comparative Example 3 were prepared using the same method as in Example 2), except that the iron oxide-containing hyaluronic acid particles in the MSC / HUVEC suspension were dispensed at 50% of the cell volume (Fig. 3d).

[0097] Meanwhile, iron oxide-containing hyaluronic acid-based cell spheroids (HA@MPS_5) of Comparative Example 4 were prepared using the same method as in Example 2), except that the iron oxide-containing hyaluronic acid particles in Example 2) were dispensed into the MSC / HUVEC suspension at 5% of the cells (Fig. 3d).

[0098] Experimental Example

[0099] Optimization of iron oxide-containing hyaluronic acid-based cell spheroids using physical stimulation

[0100] To measure the size of the physically stimulated iron oxide-containing hyaluronic acid-based MSC / HUVEC spheroids (HA@MPS) prepared through the above preparation example, the size and aspect ratio of the spheroids according to the shape and particles were measured using image j software.

[0101] Figure 3a shows the results of culture of iron oxide-containing hyaluronic acid-based MSC / HUVEC spheroids produced using MSCs and HUVECs as cells. Specifically, it was confirmed that iron oxide-containing hyaluronic acid-based MSC / HUVEC spheroids were formed 1 day after cell inoculation.

[0102] Meanwhile, Figure 3b shows the size results of iron oxide-containing hyaluronic acid-based MSC / HUVEC spheroids. One day after cell inoculation, the cell size was measured using Image J software by comparing it to the scale bar in the microscope.

[0103] As confirmed in Figure 3b, the spheroids containing hyaluronic acid particles (HA; control) measured 85.49 (±1.67) μm, the iron oxide-containing hyaluronic acid-based MSC / HUVEC spheroids (HA@MP) measured 86.88 (±1.54) μm, and the iron oxide-containing hyaluronic acid-based MSC / HUVEC spheroids using physical stimulation (HA@MPS) measured 85.55 (±1.63) μm. Combined, the average was 86.0 (±0.45) μm, showing no significant difference in size among the three groups.

[0104] Meanwhile, Figure 3c shows the aspect ratio results of iron oxide-containing hyaluronic acid-based MSC / HUVEC spheroids. To measure the spherical shape of the spheroids, the aspect ratio of the spheroids was quantitatively analyzed using the horizontal and vertical diameters of the spheroids. Specifically, HA showed 1.06 (±0.03), HA@MP showed 1.12 (±0.03), and HA@MPS showed 1.03 (±0.03). Combined, the average was 1.07 (±0.03), confirming that all three groups formed elliptical spheroids.

[0105] Meanwhile, HA10% in Fig. 3d represents a spheroid (Example 2) containing 10% by weight of hyaluronic acid particles containing iron oxide relative to the cell, and an iron oxide-containing hyaluronic acid-based MSC / HUVEC spheroid is formed, whereas HA50% in Fig. 3d represents a spheroid (Comparative Example 3) containing 50% by weight of hyaluronic acid particles containing iron oxide relative to the cell, and in this case, it was confirmed that the content of hyaluronic acid particles is excessive, so the interaction between cells is weakened and the spheroids do not clump together.

[0106] In addition, HA5% in FIG. 3d represents a spheroid (Comparative Example 4) containing hyaluronic acid particles containing iron oxide at a weight percentage of 5% relative to the cell. Although some spheroids are formed, it can be confirmed that the formation of uniform and appropriately sized spheroids is limited because the content of hyaluronic acid particles is too low.

[0107] Confirmation of the viability of iron oxide-containing hyaluronic acid-based MSC / HUVEC spheroids using physical stimulation

[0108] To check cell viability, the presence of living cells (green) and dead cells (red) in the prepared spheroids was confirmed using a LIVE / DEAD assay kit (invitrogen).

[0109] Specifically, Figure 4a shows the changes in spheroid survival rates on days 1, 3, and 7 for the three groups HA, HA@MP, and HA@MPS. It was confirmed that for all three groups, the frequency of green-stained viable cells was very high up to day 7, while the frequency of red-stained dead cells increased but did not increase.

[0110] Meanwhile, Figure 4b shows the quantified cell viability through LIVE / DEAD images, and all three groups—HA, HA@MP, and HA@MPS—showed an average cell viability of 85% by day 7, confirming that hyaluronic acid, iron oxide, and physical stimulation did not affect cell viability.

[0111] Confirmation of Enhanced Angiogenesis Ability of Iron Oxide-Containing Hyaluronic Acid-Based MSC / HUVEC Spheroids Using Physical Stimulation

[0112] Platelet endothelial cell adhesion molecule (PECAM-1) and vascular endothelial cadherin were used to evaluate the degree of angiogenesis. To confirm the enhanced angiogenesis ability of iron oxide-containing hyaluronic acid-based MSC / HUVEC spheroids using physical stimulation, spheroids were cultured for 14 days, after which the spheroids were obtained and the expression of PECAM-1 and CDH5, which can verify angiogenesis, was observed.

[0113] Figures 5a and 5c confirm the enhanced vascularization ability of iron oxide-containing hyaluronic acid-based MSC / HUVEC spheroids induced by physical stimulation using immunofluorescence staining, specifically stained with anti-PECAM-1 and CDH5. The cell nuclei appeared blue, while PECAM-1 and CDH5 appeared green. PECAM-1 and CDH5, which appeared green, showed the highest expression in iron oxide-containing hyaluronic acid-based MSC / HUVEC spheroids (HA@MPS) induced by physical stimulation, and unlike other groups, vascularization was observed even within the spheroids.

[0114] Meanwhile, Figures 5b and 5d show the results of confirming the enhanced angiogenesis ability of iron oxide-containing hyaluronic acid-based MSC / HUVEC spheroids using physical stimulation via RT-PCR; specifically, RNA was extracted using an RNA column (GenDEPOT). After measuring the total RNA concentration, cDNA was synthesized using MasterMix (Takara), and the synthesized cDNA was amplified by RT-PCR. RT-PCR was performed using QuantStudio TM It was performed using 3 (Applied Biosystems) and SYBR. The primers used are as shown in Table 1 below.

[0115] Forward(5′-3′)Reverse(5′-3′)GAPDH5′-TGACTTCAACAGCGACACC-3’5′- TTTCTGAGCCAGCCACCAG-3’CDH55′-AAGCGTGAGTCGCAAGAATG-3’5′- TCTCCAGGTTTTCGCCAGTG-3'PECAM-15′-CAAACAAATGAGGTGGCCTG-3'5′-GACTACCTGTCCTAGAGCCC-3'

[0116] The expression level of the target gene was determined using the comparative Ct method. As a result, as shown in Figure 5b, it was confirmed that on day D14, iron oxide-containing hyaluronic acid-based MSC / HUVEC spheroids (HA@MPS) treated with physical stimulation exhibited the highest PECAM-1 expression rate. Through these results, it was confirmed that physical stimulation can enhance the angiogenesis ability of MSC / HUVEC spheroids.

[0117] Confirmation of Enhanced Osteogenesis Ability of Iron Oxide-Containing Hyaluronic Acid-Based MSC / HUVEC Spheroids Using Physical Stimulation

[0118] To confirm the relationship between the enhanced vascularization ability and osteodifferentiation ability of HA@MPS spheroids, osteopontin (OPN), which is used to evaluate osteodifferentiation ability, was used. OPN is well known as a marker expressed in the late stages of osteodifferentiation.

[0119] After culturing each spheroid for 14 days, the spheroids were obtained, and the expression of OPN, which can verify bone differentiation ability, was observed.

[0120] Figure 6a shows the results of confirming the enhanced osteogenicity of iron oxide-containing hyaluronic acid-based MSC / HUVEC spheroids using physical stimulation by immunofluorescence staining, which was stained with anti-OPN. Cell nuclei appeared blue, and OPN appeared green. OPN, which appeared green, did not show expression on day 7, but it was confirmed that the highest expression was observed in iron oxide-containing hyaluronic acid-based MSC / HUVEC spheroids (HA@MPS) using physical stimulation on day 14.

[0121] Figure 6b shows the results of confirming the enhanced osteogenic differentiation ability of iron oxide-containing hyaluronic acid-based MSC / HUVEC spheroids using physical stimulation by RT-PCR, and the specific RT-PCR method is the same as the angiogenesis ability confirmation experiment above. The primers used are as shown in Table 2 below.

[0122] Forward(5′-3′)Reverse(5′-3′)GAPDH5′-TGACTTCAACAGCGACACC-3’5′- TTTCTGAGCCAGCCACCAG-3’OPN5′-GTCCACGGTCTGGTCTTCAG-3’5′- CAGCCATTGCTCAGGCAAC-3’

[0123] As a result, as shown in Figure 6b, it was confirmed that on day D14, the iron oxide-containing hyaluronic acid-based MSC / HUVEC spheroid (HA@MPS) with physical stimulation showed a higher OPN expression rate compared to the case without physical stimulation. Furthermore, it was confirmed that the spheroid (HA@MPS+) prepared in the same manner as Example 1 (HA@MPS), except that physical stimulation was repeated twice a day at the same time with a 6-hour interval, showed a superior OPN expression rate compared to the spheroid (HA@MPS) of Example 1.

[0124] Through the above results, it can be confirmed that physical stimulation can improve the osteogenic differentiation ability of MSC / HUVEC spheroids.

[0125]

[0126] Although preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements by those skilled in the art using the basic concept of the present invention as defined in the following claims also fall within the scope of the present invention.

[0127] The present invention relates to a hyaluronic acid-based cell spheroid containing iron oxide and a method for manufacturing the same.

Claims

1. Hyaluronic acid particles containing iron oxide and cells Hyaluronic acid-based cell spheroids containing iron oxide.

2. In Paragraph 1, The above cells are selected from the group consisting of endothelial cells, mesenchymal stem cells, and mixtures thereof. Hyaluronic acid-based cell spheroids containing iron oxide.

3. In Paragraph 1, The above cell spheroids have excellent bone differentiation and vascularization capabilities when physical stimulation is induced. Hyaluronic acid-based cell spheroids containing iron oxide.

4. In Paragraph 1, The aspect ratio of the cell spheroid is 1.0 to 1.

2. Hyaluronic acid-based cell spheroids containing iron oxide.

5. A step of preparing hyaluronic acid particles containing iron oxide; A step of mixing and culturing the hyaluronic acid particles containing the iron oxide above into a cell suspension to form spheroids in a culture; and A step comprising stirring the above culture to induce physical stimulation Method for manufacturing hyaluronic acid-based cell spheroids containing iron oxide.

6. In Paragraph 5, The step of inducing the above physical stimulation is to enhance the osteogenic differentiation and vascularization capabilities of cell spheroids. Method for manufacturing hyaluronic acid-based cell spheroids containing iron oxide.

7. In Paragraph 5, The step of preparing hyaluronic acid particles containing the iron oxide mentioned above is, A step of obtaining a polymer aqueous solution by mixing hyaluronic acid, iron oxide, and a crosslinking agent in a basic aqueous solution; A step of forming a w / o emulsion by mixing the above polymer aqueous solution into an oil phase in which a surfactant is dissolved; A step of stirring the above w / o emulsion to cross-link hyaluronic acid and iron oxide; A step of neutralizing the w / o emulsion that has undergone the above stirring step; and A step comprising separating hyaluronic acid particles containing iron oxide from the above w / o emulsion Method for manufacturing hyaluronic acid-based cell spheroids containing iron oxide.

8. In Paragraph 5, The crosslinking agent is selected from the group consisting of polyethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether, butylene glycol diglycidyl ether, ethylene glycol diglycidyl ether, 1,6-hexanediol diglycidyl ether, propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, polytetramethylglycol diglycidyl ether, neopentyl glycol diglycidyl ether, polyglycerol polyglycidyl ether, diglycerol polyglycidyl ether, glycerol polyglycidyl ether, trimethylpropane polyglycidyl ether, 1,2-(bis(2,3-epoxypropoxy)ethylene), pentaerythritol polyglycidyl ether, sorbitol polyglycidyl ether, and mixtures thereof. Method for manufacturing hyaluronic acid-based cell spheroids containing iron oxide.

9. In Paragraph 5, The above surfactant is selected from the group consisting of cetyl PEG / PPG-10 / 1 dimethicone (ABIL EM-90)), sorbitan sesquioleate (ARLACEL 83)), polyethylene glycol (30) dipolyhydroxy stearate (ARLACEL P135)), and mixtures thereof. Method for manufacturing hyaluronic acid-based cell spheroids containing iron oxide.

10. In Paragraph 5, The above oil phase is selected from the group consisting of cetyl ethylhexanoate, dodecane, heptane, and mixtures thereof. Method for manufacturing hyaluronic acid-based cell spheroids containing iron oxide.

11. A cell spheroid according to paragraphs 1 to 4 Composition for bone tissue regeneration.

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