Three-dimensional cell carrier for producing three-dimensional cell tissue implantable by syringe, and use thereof

Abstract

The present invention provides, as a three-dimensional scaffold, a collagen-hyaluronic acid-based microgel for producing a three-dimensional cell construct having a size enabling implantation by syringe. The three-dimensional scaffold of the present invention enables the production of three-dimensional cell tissue implantable by syringe and, furthermore, allows oxygen and nutrients to be smoothly supplied to the center of the three-dimensional cell tissue so that the survival rate of cells after culture and implantation can be increased.

Description

3D cell carrier for manufacturing 3D cell tissues transplantable by syringe and use thereof

[0001] The present invention relates to a collagen and hyaluronic acid-based microgel for producing a three-dimensional cell tissue that can be transplanted using a syringe, and a three-dimensional cell tissue produced using the same.

[0002] The number of people with peripheral artery disease (PAD) worldwide is estimated to be approximately 200 million. Critical limb ischemia (CLI) is one of the most severe clinical manifestations of atherosclerotic PAD, and approximately 10% of patients with PAD are known to develop CLI. The number of PAD patients is expected to increase due to the aging population and the rise in diabetes and smoking.

[0003] Twenty-five percent of CLI patients die within one year, and the five-year mortality rate is estimated to be over 50%. Thirty percent of CLI patients require lower extremity amputation, and approximately 150,000 patients undergo limb amputations annually. While other treatments, such as arterial bypass and percutaneous angioplasty, are available, approximately 40% of CLI patients are not suitable. While angiogenic therapy is another treatment option, gene therapies such as VEGF, FGF1, and HGF have limited efficacy and potential side effects. Therefore, stem cell therapy is emerging as a promising treatment option for angiogenic therapy.

[0004] Meanwhile, microgels are micro-sized hydrogels manufactured by chemically or physically cross-linking hydrophilic polymers to form a network structure. Typically, microgels are manufactured by dispersing an inner phase containing a hydrophilic polymer in an outer phase containing a surfactant, followed by cross-linking of the inner phase. Microgels can be manufactured in various sizes and shapes, and their physical properties, such as cross-linking density, can be controlled, making them excellent candidates for cell scaffolds. Collagen, widely used as a biocompatible polymer in hydrogels, is the most common protein found in the human body and the most abundant protein in mammals, accounting for approximately 25-35% of total protein.

[0005] The present inventors developed a paste-type three-dimensional cell tissue that can be transplanted using a syringe by providing a collagen and hyaluronic acid-based microgel as a cell carrier.

[0006] Collagen is capable of biological interaction with cells, and the present invention aims to provide a three-dimensional cell tissue made in the form of a paste using collagen microgel as a main component and capable of non-invasive injection into a living body using a syringe, and to provide a cell carrier made of collagen and hyaluronic acid, a three-dimensional cell tissue made by culturing cells together with the carrier, and a method for manufacturing each, and a pharmaceutical use thereof.

[0007] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0008] To solve the above problem, the present invention provides a microgel composition comprising 9.5% to 10.5% (w / v) collagen and 4.5% to 5.5% (w / v) hyaluronic acid.

[0009] As one embodiment of the present invention, the average size of the microgel may be 20 to 50 μm.

[0010] In addition, the present invention provides a three-dimensional scaffold manufactured using the microgel composition.

[0011] Additionally, the present invention provides a three-dimensional scaffold comprising 9.5% to 10.5% (w / v) collagen and 4.5% to 5.5% (w / v) hyaluronic acid.

[0012] As one embodiment of the present invention, the three-dimensional scaffold may be loaded with cells.

[0013] In addition, the present invention provides a three-dimensional cell structure produced by co-culturing cells with the microgel or three-dimensional scaffold.

[0014] As one embodiment of the present invention, the three-dimensional cell structure may be manufactured by mixing microgel or three-dimensional scaffold and cells at a volume ratio of 1:3 to 5.

[0015] As another embodiment of the present invention, the cell may be a stem cell, specifically an adult stem cell, and preferably an adipose-derived stem cell.

[0016] In the present invention, the three-dimensional cell structure can be injected into the human body through a syringe needle.

[0017] In addition, the present invention provides a use of the three-dimensional cell structure for treating peripheral arterial disease.

[0018] In the present invention, the three-dimensional cell structure can be manufactured in the form of an injection and provided as a pharmaceutical composition for peripheral artery treatment.

[0019] In addition, the present invention can provide a peripheral artery treatment method including a step of administering the three-dimensional cell structure to a patient with peripheral artery disease, and the three-dimensional cell structure can be used for the purpose of manufacturing an injection for treating peripheral artery disease.

[0020] As one embodiment of the present invention, the peripheral arterial disease may be severe limb ischemia.

[0021] In addition, the present invention provides a method for manufacturing a three-dimensional cell structure comprising the following steps:

[0022] (1) A step of mixing 9.5% to 10.5 (w / v) collagen and 4.5 to 5.5% (w / v) hyaluronic acid and gelling at 36 to 38°C;

[0023] (2) a step of preparing a microgel by stirring the mixture after gelation; and

[0024] (3) A step of mixing and culturing the microgel and cells.

[0025] As one embodiment of the present invention, the gelation in step (1) may be performed at 37°C.

[0026] As another embodiment of the present invention, the step (3) may be to culture the microgel and cells by mixing them in a volume ratio of 1:3 to 5.

[0027] The three-dimensional cell tissue of the present invention has the advantageous effect of being provided in a size small enough to be transplanted using a syringe, providing a structure with pores that allow smooth supply of oxygen and nutrients to the center, and maintaining a high cell survival rate through this.

[0028] Figure 1 is a schematic diagram of a paste-type 3D cell tissue manufacturing process of the present invention.

[0029] Figure 2 illustrates the formation of a composite preparation by biological cross-linking of collagen microgels and cells.

[0030] Figure 3 illustrates the biological cross-linking mechanism between collagen microgels and cells.

[0031] Figures 4a to 4d show the results of confirming the particle characteristics of collagen-hyaluronic acid microgels according to the hyaluronic acid content. Specifically, Figure 4a is a SEM image showing the morphology of hydrogels (ae) and microgels (fj) composed of 10% collagen and 0%, 0.5%, 1%, 2.5%, or 5% HA, and a super-resolution STORM image of the microgel (ko). Figure 4b is a graph quantifying the microgel fiber thickness based on a 3D super-resolution STORM image, and Figures 4c and 4d are graphs showing the frequency size distribution of microgels according to the length and width measured using Morphologi G3.

[0032] Figure 5 shows the results of confirming the characteristics of cell tissue according to the cell and CMG ratio. Specifically, Figure 5A is a photograph showing the injectability of 3D structures and CMG manufactured at ratios of 1:1, 1:2, and 1:4 through a 24-gauge needle (scale bar = 5 mm). Figure 5B is a graph showing the storage (solid circle) and loss (open circle) coefficients of CMG or 3D structures. Figure 5C is a fluorescent image showing the distribution of cells and collagen type 1dml within the 3D structures. Nuclei were counterstained with DAPI, and the scale bar represents 50 μm. Figure 5D is a color SEM image showing the distribution of CMG (yellow) and cells (pink) within 3D structures manufactured at different cell:CMG ratios (scale bar = 20 μm).

[0033] Figure 6 shows the results of confirming the characteristics of 3D cell tissues produced by collagen-hyaluronic acid and cell co-culture. Specifically, Figure 6A is a photograph showing the effect of CD44 or integrin β1 blockade on 3D structure formation (scale bar = 2 mm). Figure 6B is a photograph showing the effect of chemical inhibitors on 3D structure formation (scale bar = 2 mm). Figure 6C is a graph quantifying the effect of function-blocking antibodies or chemical inhibitors on the size of 3D structures (n = 3). Figure 6D is a Western blot showing the level of phosphorylated FAK based on the cell:CMG ratio (pellet size). Figure 6E is a graph showing the effect of function blockade of CD44 or integrin β1 on cell viability determined by trypan blue exclusion assay (n = 3). Figure 6F is a schematic diagram of how cells assemble into CMGs to form 3D structures through biological cross-linking with integrin and signaling pathway activation, and how treatment with function-blocking antibodies or chemical inhibitors inhibits 3D formation.

[0034] Figure 7 shows the results of confirming the pores of the cell tissue and the degree of material transfer into the cell tissue according to the cell and CMG ratio. Specifically, Figure 7A is a Micro-CT visualization image of a cross-section of a 3D structure prepared at a ratio of 1:0, 1:1, 1:2, and 1:4 (cells:CMG, pellet size), where the pores are indicated in gray and CMG or cells are indicated in white. Figure 7B is a fluorescence image showing the cellular uptake of 2-NBD-glucose into the interior of the 3D structure. Figure 7C is a fluorescence image stained for pimonidazole, a hypoxia marker. Figure 7D is a fluorescence image of a 3D structure prepared at a ratio of 1:0, 1:2, 1:2, or 1:4 (2×10 5These are images of live and dead cells stained in 3D structures fabricated with hASCs / sample, cell:CMG ratios by pellet size. The scale bar in Figs. 7A to 7D represents 500 μm. Fig. 7E is a graph quantifying the porosity within the 3D structures analyzed using CTAn software, and Fig. 7F is a graph quantifying the intracellular ATP level measured using the Cell Titer-Glo® kit. Fig. 7G is a Western blot of cell lysates prepared from hASCs cultured in monolayer and 3D structures fabricated at ratios of 1:0, 1:1, 1:2, and 1:4 (cell:CMG, pellet size) stained with pimonidazole antibody. Figure 7H is a graph showing cell viability over time in 3D structures manufactured at ratios of 1:0, 1:1, 1:2, and 1:4 (cells:CMG, pellet size).

[0035] Figure 8a shows the results of differentially expressed genes in a cell tissue produced with a cell and CMG ratio of 1:4 compared to a cell tissue produced solely with cells, and Figure 8b shows the results of an additional heatmap analysis of Figure 8a. Figure 8c shows the results of differentially expressed genes in a cell and CMG ratio of 1:0, 1:1, 1:2, and 1:4 (2 x 10 5 The mRNA and protein expression levels of VEGF, IL-8, and TIMP1 in 3D structures fabricated with hASCs / sample (cell:CMG ratio by pellet size) were confirmed by RT-PCR and ELISA.

[0036] Figure 9 shows the results of evaluating the angiogenic ability of the 3D structure in vitro and ex vivo. Specifically, Figure 9 A shows the angiogenic ability of the 3D structure in 1:0, 1:1, 1:2, and 1:4 ratios (2 x 10) with HUVEC cells labeled with green fluorescent protein. 5This is an image after 16 hours of co-culture of a 3D structure fabricated with hASCs / sample, cell:CMG ratio by pellet size (scale bar=200μm), and Fig. 9B is a graph quantifying this. Endothelial basal media (EBM) and CMG treatment alone were used as negative controls, and Endothelial growth media (EGM: EBM + supplements) was used as a positive control. Fig. 9C is an image confirming microvessel sprouting on the 7th day after co-culture of the 3D structure with an aortic ring, and Fig. 9D is a graph quantifying this.

[0037] Figure 10 shows the results of 3D cell tissue administration to an animal model of severe limb ischemia. Mice were surgically induced to ischemia in the left leg, and on the first day after surgery, each experimental substance (CMG alone, CMG / hASC suspension, cell aggregates, or 3D structure of CMG / hASC; 2x10 5 hASCs / mouse, hASC:CMG=1:4) were injected. Figure 10 A is a representative image of the hindlimb morphology and blood perfusion of the CLI mouse model on days 0, 7, 14, and 28 after surgery, Figure 10 B is the quantification of the blood perfusion ratio, and Figure 10 C is the result of the physiological status analysis of the ischemic limb on day 28 after surgery.

[0038] Figure 11 shows the effect of 3D cell construct administration on the survival and therapeutic effect of ischemic muscles. Specifically, ischemic thigh muscles collected from mice administered CMG / hASCs suspension, cell aggregates, or 3D constructs of CMG-hASCs were immunofluorescently stained for HNA (Figure 11a), laminin (Figure 11b), or CD31 and HNA (Figure 11c). DAPI was used for nuclear staining, and the scale bar represents 50 μm. Figure 11d shows the quantification of HNA-positive cells, and Figure 11e shows the results in mm.2 Quantification of the number of muscle fibers with at least one central nucleus, and Figure 11f quantification of CD31 positive cells in ischemic thigh muscles.

[0039] Figure 12 shows the particle size according to the hyaluronic acid concentration, specifically, the frequency distribution of the length (C) and width (D) of the microgel particles manufactured using 10% collagen + HA (0%-5%) (A) and HA (2.5%) + collagen (5%-15%) (B).

[0040] In Fig. 13, A and B are graphs that confirm and quantify the effect of HA changes on the 3D cell structure using digital photographs, 2*10 5 The effect of hyaluronic acid concentration on cell tissues produced by mixing hASC cells at a cell:CMG ratio of 1:4 was confirmed. One-way ANOVA analysis was followed by a multiple comparison test (ns, not significant, *p<0.05). C and D show rheological properties showing storage (solid circle) and loss (open circle) moduli according to changes in hyaluronic acid concentration.

[0041] Figure 14 is an immunofluorescence staining photograph confirming the degree of cell dispersion according to various hyaluronic acid concentrations in a 3D cell tissue.

[0042] Figure 15 demonstrates the influence of collagen concentration on 3D cell tissues. Specifically, A and B are photographs of cell tissues fabricated with a mixture of 2.5% hyaluronic acid and 5 to 15% collagen, along with graphs showing their diameters. C and D demonstrate rheological properties, demonstrating the storage (solid circle) and loss (open circle) moduli according to varying collagen content.

[0043] Figure 16 is an immunofluorescence staining photograph confirming the degree of cell dispersion according to various collagen concentrations in a 3D cell tissue.

[0044] Figure 17 shows the results of immunofluorescence staining that confirmed cell viability according to collagen concentration in a 3D cell tissue (scale bar=500um).

[0045] The present invention relates to a cell-loaded microgel that can be administered by a syringe, wherein the microgel of the present invention comprises 9.5% to 10.5 (w / v) collagen and 4.5 to 5.5% (w / v) hyaluronic acid, and preferably has an average size of 20 to 50 μm.

[0046] In this specification, "hydrogel" may refer to a three-dimensional network structure made by cross-linking hydrophilic polymers through covalent or non-covalent bonds. Due to the hydrophilicity of the constituent materials, it has the property of absorbing and swelling a large amount of water in an aqueous solution and in an aqueous environment, but does not dissolve due to the cross-linked structure. Therefore, hydrogels with various shapes and properties can be made depending on the constituent materials and manufacturing method, and since they generally contain a large amount of water, they may have properties intermediate between liquid and solid. Among these, "microgel" refers to a micro-sized hydrogel manufactured by chemically or physically cross-linking hydrophilic polymers to build a network structure.

[0047] As used herein, “collagen” refers to the most common protein found in the human body, the most abundant protein in mammals, and accounting for approximately 25-35% of total proteins. In particular, it is a major component of bones, tendons, and ligaments, and is known to primarily play a role in maintaining the structure of organs. It can be easily extracted from the skin of cows or pigs. In addition to pure collagen, the collagen may include collagen derivatives. With respect to collagen, its type (source) is not particularly limited, and for example, various collagens derived from mammals, fish, such as cow bones, cow skin, pig bones, and pig skin, can be used. In addition, the collagen may have a molecular weight of 100,000 to 250,000, 120,000 to 240,000, or 150,000 to 200,000.

[0048] In this specification, “hyaluronic acid” means “hyaluronan”, “hyaluronate”,

[0049] (hyaluronate)" or "HA" and may include the following chemical formula 1 or a pharmaceutically acceptable salt thereof, for example, sodium hyaluronate (NaHA), potassium hyaluronate, magnesium hyaluronate, potassium hyaluronate and combinations thereof.

[0050]

[0051] In the above formula 1, n represents the number of repeating units. Hyaluronic acid of any origin, including bacterial and algal origin, is useful. Useful hyaluronic acid may have a molecular weight range of about 0.1 MDa to about 6.0 MDa, for example, about 1.5 MDa to about 6.0 MDa, about 2.5 MDa to about 6.0 MDa, about 3.5 MDa to about 6.0 MDa, about 0.1 MDa to about 5.0 MDa, about 0.1 MDa to about 4.0 MDa, or about 0.1 MDa to about 3.0 MDa.

[0052] Accordingly, in one embodiment, the microgel, the composition comprising the microgel, and the three-dimensional scaffold may each substantially contain no additional cross-linking agent.

[0053] As used herein, the terms "does not contain," "is free of," and "substantially free of" may mean that the cross-linking agent, e.g., a chemical cross-linking agent, is not included in an amount insufficient to fully perform its role, i.e., not in an effective amount. Specifically, it may mean less than about 1 wt %, less than 0.5 wt %, less than 0.1 wt %, and in some cases, less than about 0.05 wt %, and in other cases, none, of the total composition.

[0054] In the present invention, the microgel may be loaded with cells. In addition, the microgel may further contain a growth factor or a differentiation factor. The cells may include cells to be cultured in the microgel composition, cells to be differentiated into tissues or other cells, or cells to be used for tissue regeneration. Examples of the cells may be stem cells, sensory cells, brain cells, germ cells, epithelial cells, immune cells, cancer cells, or a combination thereof. The stem cells may refer to cells having differentiation potential, and the cells having differentiation potential may include, for example, blast cells, hepatocytes, fibroblasts, myoblasts, adult stem cells, mesenchymal stem cells, adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, nerve-derived mesenchymal stem cells, placental-derived mesenchymal stem cells, or umbilical cord blood stem cells, or a combination thereof. The growth factor may refer to a substance capable of regulating the growth and function of cells. The above differentiation factor may refer to a substance that induces differentiation of cells into tissues or into other cells. For example, the growth factor or differentiation factor may include transforming growth factor (TGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), platelet-derived endothelial growth factor (PDGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), cytokines, chemokines, or a combination thereof.

[0055] In the present invention, the microgel can be provided for use in loading cells.

[0056] In this specification, a microgel having a structure in which cells can be loaded is referred to as a cell carrier. At this time, the cell carrier preferably has a three-dimensional structure so that cells cultured on the microgel can have a three-dimensional structure. In this specification, the cell carrier having the three-dimensional structure is referred to as a three-dimensional scaffold.

[0057] In the present invention, it is important that the microgel be manufactured to an appropriate size so that it can be loaded with cells and administered into a living body via a syringe. In the present invention, the average diameter of the three-dimensional cell carrier and three-dimensional scaffold is preferably 20 to 50 μm.

[0058] In the present invention, cells cultured in a three-dimensional scaffold form a cell structure having a three-dimensional structure, and the cell structure in the present specification is expressed as a three-dimensional cell structure, a three-dimensional cell tissue, a 3D structure, etc. The cell structure is preferably cultured by mixing microgel and cells in a volume ratio of 1:3 to 5, preferably 1:4, and the average diameter of the three-dimensional cell structure is preferably 2 to 3 mm. The present invention confirmed that transplantation of the above-described three-dimensional cell structure is possible through a 24-gauge needle.

[0059] In the present invention, the microgel comprises the following steps:

[0060] (1) a step of reacting 9.5% to 10.5% (w / v) collagen and 4.5% to 5.5% (w / v) hyaluronic acid to form an ionic-bonded collagen-hyaluronic acid complex; (2) a step of homogenizing the collagen-hyaluronic acid complex to prepare a dispersion; and (3) a step of naturally sedimenting the dispersion.

[0061] The above ionic bonded collagen-hyaluronic acid complex is in the form of a gel.

[0062] In addition, the method for manufacturing a three-dimensional cell structure in the present invention includes the following steps:

[0063] (1) The step of manufacturing the microgel described above; and (2) the step of mixing the microgel and cells and culturing them.

[0064] As one embodiment of the present invention, the microgel and cells can be cultured by mixing them in a volume ratio of 1:3 to 5, preferably 1:4.

[0065] Meanwhile, the present invention demonstrated a high level of therapeutic efficacy, along with increased blood flow, when a 3D cell construct manufactured using adipose-derived stem cells was administered intramuscularly to CLI-induced mice. The administration of the 3D cell construct according to the present invention demonstrated a high level of therapeutic efficacy despite administering approximately 10 times fewer cells than those typically administered in animal experiments to confirm therapeutic efficacy.

[0066] Accordingly, the present invention provides a three-dimensional cell structure formed by mixing and culturing stem cells and the microgel of the present invention for use in the treatment of peripheral arterial disease, such as severe limb ischemia.

[0067] The present invention provides a pharmaceutical composition for treating peripheral arterial disease comprising the three-dimensional cell structure as an active ingredient.

[0068] As described above, the three-dimensional cell structure included in the pharmaceutical composition of the present invention preferably has an average diameter of about 2 to 3 mm.

[0069] In addition, the pharmaceutical composition of the present invention can be formulated as a sterile aqueous solution, non-aqueous solvent, or suspension for parenteral administration, and specifically, can be formulated as an injection.

[0070] In this specification, “treatment” means any action to alleviate the symptoms of peripheral arterial disease, “subject” means a mammal diagnosed with peripheral arterial disease, and “administration” means parenteral administration, specifically intravenous administration, intramuscular administration, subcutaneous administration, etc.

[0071] The present invention is susceptible to various modifications and embodiments. Specific embodiments are illustrated in the drawings and described in detail in the following detailed description. However, this is not intended to limit the present invention to specific embodiments, but rather to encompass all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention. In describing the present invention, detailed descriptions of related known technologies will be omitted if they are deemed to obscure the gist of the present invention.

[0072] [Experimental Methods and Materials]

[0073] 1. Collagen / HA microgel manufacturing

[0074] 25% (w / w) atelocollagen (MSBio, Inc., Seoul, South Korea) and 10% (w / w) sodium hyaluronate (HA) (Contipro Inc., Dolni Dobrou , Czech Republic) was prepared by dissolving atelocollagen in 0.01 N hydrochloric acid and HA in phosphate-buffered saline (PBS). Collagen and HA were placed in a tube and mixed on ice. The pH of the mixture was adjusted to pH 7.1–7.3 by adding 2 N NaOH or 1 N HCl. To obtain the desired final volume, PBS was added, and the mixture was incubated at 37°C for at least 30 min to allow gelation. The collagen / HA gel was added to a new tube containing StemPro™ serum-free medium (SFM, Gibco, Waltham, MA, USA) and glass beads. The gel was broken into microgels by shaking at 1500 rpm for 30 s using a bead beater (Mini-Beadbeater-24, BioSpec Products, Bartlesville, OK, USA). The generated CMG (collagen microgel) solution was vacuum filtered using a 100 μm filter and stored at 25 ± 2°C until use.

[0075]

[0076] 2. Characteristic Analysis

[0077] 2-1. Zeta potential analysis

[0078] Hydrogels containing various concentrations of collagen and HA were prepared and ground in distilled water at a pH value of 3–11. The surface charge of CMG was measured by measuring the zeta potential of the microgels using a Zetasizer Nano ZS system (Malvern Panalytical Ltd., Nottingham, UK).

[0079]

[0080] 2-2. Scanning electron microscopy

[0081] Samples were fixed with 2.5% glutaraldehyde (Sigma-Aldrich, St. Louis, MO, USA) for primary fixation for 2 h, followed by secondary fixation with 2% osmium tetroxide (Sigma-Aldrich) for 3 h at 25 ± 2 °C. The fixed samples were washed with distilled water and dehydrated with increasing concentrations of ethanol (30%, 50%, 70%, 80%, 90%, and 100%) for 5 min each at 25 ± 2 °C. Hexamethyldisilazane (Sigma-Aldrich) was added, and the samples were dried overnight in a vacuum chamber at 25 ± 2 °C. The samples were placed on SEM stubs and sputter-coated with platinum, vertically and on both sides for 60 s each. Images of the prepared samples were captured using a field-emission SEM (Inspect F50, FEI Company, Hillsboro, OR, USA).

[0082]

[0083] 2-3. 3D stochastic optical reconstruction microscopy (STORM) images

[0084] Collagen fiber thickness of CMG was analyzed using 3D stochastic optical reconstruction microscopy (STORM). CMG was first conjugated to Alexa FluorTM 647 by incubating with NHS-ester-Alexa-647 at 5 μg / mL for 1 h at 25 ± 2°C. CMG was then washed in PBS to remove unbound dye, mounted on #1.5H coverslips precoated with 0.5% 3-aminopropyltriethoxysilane, and imaged in STORM imaging buffer (100 mM cysteamine, 10% glucose, 0.8 mg / mL glucose oxidase, 40 μg / mL catalase, 50 mM Tris-HCl, and 10 mM NaCl in PBS, pH 8.0). Samples were sealed with nail polish before imaging.

[0085] The prepared sample was loaded into a home-built STORM setup (objective: 100× / 1.45) to capture the astigmatic point spread function of Alexa FluorTM 647 molecules for 3D localization. Single-molecule videos of CMG were captured with a weak 405 nm laser excitation (average illumination intensity: ~10 kW / cm). 2 ) were acquired at a rate of 50 frames / s over 20,000 image frames using a 642 nm laser. The STORM video data were then localized and 3D STORM images were reconstructed. The lateral and axial cross-sectional intensity profiles of 10 random fibers within the images were analyzed, and the cross-sectional thickness was determined as the full width at the half-maximum intensity of these profiles.

[0086]

[0087] 2-4. Size distribution of CMG

[0088] The size distribution of CMGs was analyzed using an automated Mophologi G3 optical microscope (Malvern Panalytical Ltd.). Specifically, microgels were dispersed in distilled water on glass slides, covered with a coverslip, and then scanned using an automated microscope to capture images of CMGs. The images were analyzed using a computer equipped with Morphologi software (Malvern Panalytical Ltd.).

[0089]

[0090] 3. CMG as a stem cell carrier

[0091] Human adipose-derived stem cells (hASC) were obtained from S. Biomedics (Seoul, South Korea) and were confirmed to meet the minimum standards set by the International Society for Cell Therapy. hASC were cultured in CEFOgro medium (CEFO Co., Seoul, South Korea) for up to 5 passages in a humidified chamber set at 37°C and 5% CO2. To prepare 3D constructs, hASC were trypsinized, resuspended in SFM medium, and seeded at 2 × 10 per well in 96-well round-bottom ultra-low attachment plates. 5 Cells were seeded. The prepared CMG solution was added to each well at a ratio of 1:0, 1:1, 1:2, or 1:4 (cell:CMG ratio predetermined by pellet volume), and SFM was added to make up a total volume of 300 μL per well. The resulting cell-CMG culture was mixed well by pipetting and stored in an incubator at 37ºC for 24 h to form 3D structures.

[0092]

[0093] 4. Rheological properties of 3D structures

[0094] To form a 3D structure with a volume of approximately 500 μL, 8 x 10 6 cells, 4 x 10 6 cells or 2 x 10 6 Cells were mixed with CMG at a ratio of 1:1, 1:2, or 1:4 in a non-tissue cultured 24-well plate and cultured at 37°C for 24 h. The storage (G′) and loss (G″) moduli were measured to evaluate the viscoelastic properties of the 3D structures using an MCR 102 rheometer (Anton-Paar, Graz, Austria). Dynamic time sweeps were performed at a frequency of 10 rad / s and 0.5% strain at 25°C until a plateau was reached.

[0095]

[0096] 5. Function-blocking assay

[0097] To block the function of CD44 or integrin β1 receptor before forming 3D structures, the harvested cells were treated with rat anti-CD44 (Hermes-1, Invitrogen, Waltham, MA, USA) at 0, 10, 30, or 50 μg / mL or rat anti-human CD29 (integrin β1, BD Biosciences, Franklin Lakes, NJ, USA) at 0, 5, 10, or 20 μg / mL and pre-incubated for 30 min in a 37°C incubator. Then, they were mixed with CMG at a ratio of 1:0, 1:1, 1:2, or 1:4 and incubated at 37°C for 24 h to form 3D structures. For inhibitor treatment, cells were trypsinized, harvested, and treated with 0, 10, 30, or 50 μM DMSO or focal adhesion kinase (FAK; PF-573228), Rho-associated coiled-coil kinase (ROCK; Y-27632), and myosin (blebbistatin). The cells were then mixed with CMG at a ratio of 1:0, 1:1, 1:2, or 1:4 and incubated at 37°C for 24 h to form 3D structures. As a negative control, 50 μM extracellular signal-regulated kinase inhibitor (ERK; U0126) was used. Inhibition of 3D cell formation was determined by measuring the diameter of the final 3D structures. Viability was assessed using a trypan blue exclusion assay.

[0098]

[0099] 6. Characterization of 3D structures

[0100] 6-1. Micro-CT

[0101] The porosity of the 3D structures was analyzed using micro-CT. Specifically, the samples were fixed with 4% paraformaldehyde and stained with 5% phosphotungstic acid in 70% ethanol for 3 days. Then, the prepared samples were placed in tubes containing phosphate-buffered saline (PBS), and computed tomography scans were performed using micro-CT (Skyscan 1172, Bruker, Billerica, MA, USA) (parameters: aluminum filter, 0.5 mm; X-ray tube voltage, 75 kV; tube current, 134 μA; pixel size, 2.6 μm; scanning angle range, 360°). The data were reconstructed using NRecon software (Bruker) and analyzed using CTAn software (Bruker).

[0102]

[0103] 6-2. Glucose absorption analysis

[0104] Glucose uptake assays were used to assess the mass transfer of the 3D structures. Samples were incubated with 500 μM 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) for 1 h and frozen in liquid nitrogen. Cryosections (10 μm thick) were washed in PBS for 30 s and mounted with mounting medium containing 4′,6-diamidino-2-henylindole (DAPI). Sections were visualized with an LSM 700 confocal microscope (Zeiss, Oberkochen, Germany).

[0105]

[0106] 6-3. Hypoxia

[0107] 3D constructs were prepared as described above and incubated with 10 mM pimonidazole (Hypoxyprobe, Burlington, MA, USA) for 5 h. Samples were prepared as frozen blocks for pimonidazole immunofluorescence staining or as cell lysates for quantification using Western blotting.

[0108] .

[0109]

[0110] 7. Immunofluorescence staining

[0111] For immunofluorescence staining of 3D structures, samples were prepared as frozen blocks and cut into 10-μm-thick sections. For immunofluorescence staining of thigh muscle tissue, samples were prepared as paraffin blocks and cut into 4-μm-thick sections. The prepared sections were incubated in blocking solution [2% bovine serum albumin (BSA) + 0.1% Triton X-100 in PBS] for 1 h. The sections were incubated with primary antibodies (diluted in blocking solution) overnight at 4°C and then incubated with fluorescence-conjugated secondary antibodies (diluted 1:500 in blocking solution) for 1 h at 25 ± 2°C. The samples were then mounted using Vectashield antifade mounting medium containing DAPI (Vector Laboratories Inc., Newark, CA, USA), and fluorescent images were captured using a Zeiss LSM 700 confocal microscope (Zeiss).

[0112]

[0113] 8. Western blot

[0114] Cell lysates were homogenized using a Mini-Beadbeater-24 (Biospec Products) in 20 mM Tris-HCl buffer (pH 7.2) containing a cocktail of protease and phosphatase inhibitors. Cell debris and CMG were removed by centrifugation. Proteins were separated by electrophoresis on a 4–15% gradient gel (Bio-Rad Laboratories, Hercules, CA, USA) and transferred to polyvinylidene fluoride membranes (Millipore, Burlington, MA, USA). The membranes were blocked for 1 h in 5% BSA-TBST (1 M Tris-HCl, pH 7.4, 0.9% NaCl, and 0.05% Tween-20), treated with primary antibodies, diluted in 10% BSA-TBST, and incubated overnight at 4°C with shaking. The membrane was washed three times in TBST and incubated with HRP-conjugated secondary antibody diluted 1:2000 for 2 hours. Western blots were visualized using an enhanced chemiluminescence system, and images were captured using an iBright CL1500 imaging system (Thermo Fisher Scientific).

[0115]

[0116] 9. Evaluation of cell viability of 3D structures

[0117] To measure cell viability, 3D structures were stained with 20 μM of a living dye (calcein AM) and 10 μM of a dead dye (propidium iodide) for 5 h at 37°C. Samples were prepared using optimal cutting temperature compound, mounted, and coverslipped for 10-μm-thick cryosections. Fluorescence images were captured using an LSM 700 confocal microscope (Zeiss).

[0118]

[0119] 10. RNA sequence analysis

[0120] RNA sequencing analysis was performed on 3D constructs cultured with hASCs and CMGs in non-tissue culture plates at ratios of 1:0 and 1:4 for 24 h. Total mRNA was extracted using the TRIzol method and quality assessed using an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). cDNA libraries were constructed using the QuantSeq 3′ mRNA-Seq Library Prep Kit (Lexogen Inc., Vienna, Austria) according to the manufacturer's instructions. High-throughput sequencing was performed using NextSeq 500 (Illumina, San Diego, CA, USA). Sequenced QuantSeq 3′ mRNA-Seq reads were aligned using Bowtie2. Differentially expressed genes were identified based on a fold change ≥ 1.5 and a p-value < 0.05. Gene classification was based on searches performed using the DAVID and Medline search engines.

[0121]

[0122] 11. In vitro tube formation assay

[0123] Human umbilical vein endothelial cells (HUVECs) labeled with green fluorescent protein were cultured in EGM-2 medium (Lonza, Basel, Switzerland) supplemented with supplements and antibiotics up to the fourth passage. For tube formation assays, 24-well transwell culture plates (SPL Life Sciences, Pocheon, South Korea) were coated with growth factor reduced-basement membrane extract (Matrigel®, Corning, Corning, NY, USA) and incubated at 37°C for 40 min. Serum-starved HUVECs were seeded at 1 × 10 per well in the lower compartment of the transwell plates. 5 The cells were sown at a cell density of 10 cells and cultured in an incubator at 37°C for 40 minutes.

[0124] Subsequently, (1) positive control, (2) negative control, (3) CMG, (4) 1:0 (cells:CMG), (5) 1:1 (cells:CMG), (6) 1:2 (cells:CMG), or (7) 1:4 (cells:CMG) were placed in the upper compartment of the transwell plate and co-cultured with HUVECs for 16 h. All experimental groups except the positive and negative controls were cultured in SFM. The positive control group was cultured in EGM supplemented with angiogenic growth factors, and the negative control group was cultured in endothelial basal medium (EBM) without additional supplements. The CMG group involved co-culturing HUVECs with CMG and was included to confirm the effect of CMG alone. Tube formation in the wells was visualized using a confocal microscope.

[0125]

[0126] 12. CLI model

[0127] 12-1. Animal Management

[0128] All animals were cared for according to the guidelines established by the National Institutes of Health. Thirty male BALB / c nu / nu mice (5 weeks old) were purchased from Orient Bio Inc. (Seongnam, South Korea). Five mice were used for the ex vivo aortic ring assay, and 25 mice were induced with CLI. All mice had free access to food and water and were acclimated for at least 1 week before use. During the experiment, mice were housed in an environment with a 12-h light / dark cycle, at 24°C, and 60% humidity. All animal procedures were approved by the International Animal Care and Use Committee of the Korea Advanced Institute of Science and Technology [KIST-2021-090].

[0129]

[0130] 12-2. Ex vivo mouse aortic ring assay

[0131] Six-week-old mice were euthanized, the thoracic cavity was opened, and the thoracic aorta was excised and carefully removed of fibroadipose tissue. The aortic tissue was cut into 1-mm aortic rings and serum-starved overnight. For ex vivo mouse aortic ring analysis, aortic rings were embedded in 1% rat tail collagen gel (Corning) in the lower compartment of a prechilled 24-well Transwell culture plate (SPL Life Sciences), and (1) positive control, (2) negative control, (3) CMG, (4) 1:0, (5) 1:1, (6) 1:2, or (7) 1:4 were placed in the upper compartment. All experimental groups except the positive and negative controls were cultured in SFM. The positive control group was cultured in EGM supplemented with angiogenic growth factors, and the negative control group was cultured in EBM. Aortic rings were co-cultured for 7 days at 37°C and 5% CO2, with media changed daily. For selective visualization of neovascularization, aortic rings were fixed and blocked with 4% PFA (1% BSA + 0.5% Triton X-100) and stained with Alexa Fluor conjugated with isolectin IB4, an endothelial cell marker. TM 488 (Invitrogen). Images were acquired using a fluorescence microscope (Zeiss). Images were analyzed using ImageJ software (ImageJ, Bethesda, MD, USA) to quantify microangiogenesis.

[0132]

[0133] 12-3. CLI induction

[0134] Twenty-five 6-week-old male BALB / c nu / nu mice were anesthetized with isoflurane. To induce ischemia, the right hind limb skin of each mouse was resected to expose the femoral artery, which was ligated with a 5-0 black silk suture (AILEE Co. Ltd., Busan, South Korea) at the proximal end of the external iliac artery and the distal end where it branches into the saphenous and popliteal arteries. The ligated femoral artery was resected, and the skin incision site was sutured. Blood flow was monitored by Laser Doppler Perfusion Imaging (Moor Instruments Ltd., Devon, UK). The mice were randomly divided into five groups (n = 5 per group) and treated with five different treatments for 1 day after surgery: (1) PBS as a negative control, (2) CMG, and (3) CMG / hASC (2 × 10 5 (4) cell / mouse suspension, (5) cell aggregates, and (6) CMG-hASC constructs. To eliminate the effects that may arise from volume differences, low dead volume syringes equipped with 24-gauge needles (Kovax-Needle, Korea Vaccine, Ansan, South Korea) were loaded with PBS to a final volume of 100 μL and administered intramuscularly into the inner thigh of the ischemic limb. Blood flow and recovery in the ischemic limb were monitored once a week for up to 28 days using Laser Doppler Perfusion Imaging.

[0135]

[0136] 12-4. In vitro and in vivo biodegradation analysis

[0137] To measure enzymatic biodegradation rates in vitro, CMG or 3D structures (cell:CMG ratios of 1:1, 1:2, and 1:4) were freeze-dried and treated with 30 U of collagenase and 10 U of hyaluronidase. The biodegradation rate was calculated by dividing the weight at a specific time point by the initial weight at 0 h and multiplying by 100.

[0138] To measure biodegradation rates in vivo, CMG conjugated to atto-488-NHS was injected into normal and ischemic limbs, and fluorescence intensity was measured using an in vivo imaging system (IVIS, Perkin Elmer, Waltham, MA, USA). Biodegradation rates were calculated by normalizing fluorescence intensities to the 0d value.

[0139]

[0140] 13. Statistical Analysis

[0141] All statistical analyses were performed using Prism 7 software (GraphPad, San Diego, CA, USA). Comparisons between multiple experimental groups were performed using one-way or two-way analysis of variance, assuming Gaussian distributions and equal standard deviations (SDs), followed by multiple comparison tests at a 95% confidence level. Statistical significance was set at p < 0.05.

[0142]

[0143] [Experimental Results]

[0144] <Example 1: Confirmation of particle characteristics according to hyaluronic acid content>

[0145] To determine whether collagen and HA interact electrostatically, CMGs composed of various concentrations of collagen (5% - 15%) or / and HA (0% - 5%) were prepared and the zeta potentials were measured in aqueous solutions over the pH range of 3-11. The isoelectric point of a 10% collagen gel was determined to be approximately 10.4. The addition of 0.5% HA to the collagen decreased the isoelectric point to approximately 4.4, and the decrease continued with increasing HA concentration. This indicates that positively charged collagen electrostatically interacts with negatively charged HA at physiological pH to form a polyionic complex.

[0146] To determine the effect of HA on collagen fibrogenesis, we mixed 10% collagen gel with various concentrations of HA (0%–5%) and investigated the thickness of the resulting collagen fibers. As shown in the SEM images of the hydrogels and the 3D super-resolution STORM images of the microgels (Figs. 4a–4b), increasing HA concentration resulted in thinner collagen fibers. When mechanical stress was applied to the hydrogels, the bulk hydrogel fragmented into micro-sized gels, and the overall size decreased with increasing HA concentration, presumably due to the thinning of the collagen fibers by HA (Fig. 4a). In contrast, when 2.5% HA was mixed with various concentrations of collagen (5%–15%), thicker collagen fibers and an increase in the overall size of the microgels were observed. Microgels prepared with 10% collagen and 5% HA exhibited the smallest and most uniform microgel size distribution and were produced with the highest yield (Figures 4c - 4d). When microgels were formed with 10% collagen and 5% HA, the smallest size was observed, with an average size of 43 μm in length and 23 μm in width. Hereinafter, microgels prepared under conditions of 10% collagen and 5% HA were used.

[0147]

[0148] <Example 2: Confirmation of cell tissue characteristics according to cell and CMG ratio>

[0149] The formation of 3D structures was determined by incubating manufactured CMGs with hASCs at ratios of 1:0, 1:1, 1:2, 1:4, 1:8, and 1:16. Increasing the cell:CMG ratio from 1:1 to 1:16 resulted in the contraction of the CMGs by the cells, forming 3D structures. However, the degree of contraction decreased with increasing cell:CMG ratios. Furthermore, when each group was removed from the medium, the 1:0, 1:1, 1:2, and 1:4 groups were able to maintain structural integrity, whereas the 1:8 and 1:16 groups failed to maintain structural integrity and collapsed.

[0150] To assess the injectability of the 3D constructs, each group was passed through a 24-gauge needle, and all experimental groups were confirmed to pass through the needle without affecting cell viability (Fig. 5A). Unlike the 3D constructs containing hASCs and CMGs, the CMG-only experimental group required the presence of hASCs to drop out of the needle and connect the microgels.

[0151] Analysis of the rheological properties of the experimental groups showed that when 0.5% strain was applied, the mechanical properties of CMG alone were weak at approximately 100 Pa and 60 Pa, respectively, but when hASCs were incubated with CMG, the viscous and elastic properties increased due to cell cross-linking with the microgels (Fig. 5B). The distribution of hASCs and CMG in the 3D constructs was observed through immunofluorescence and SEM images of the cross-sections of the 3D constructs. The results showed that the cells and CMG were uniformly distributed throughout the 3D constructs. Although CMG was not included in the 1:0 group, collagen type 1 was still observed, which seems to be collagen type 1 secreted from hASCs (Fig. 5C and D). Increasing the CMG ratio in the 3D constructs increased the distance between adjacent cells.

[0152]

[0153] <Example 3: Confirmation of the mechanism of 3D cell tissue formation>

[0154] To determine the mechanism by which hASCs and CMGs assemble into 3D structures, we used antibodies and inhibitors to block signaling pathways associated with actin contraction. Since the manufactured CMGs are composed of HA and collagen, we used function-blocking antibodies to block CD44 (the HA receptor) or integrin β1 (the collagen receptor) on the cell membrane, and then cultured them with CMGs to determine whether blocking these receptors affected 3D formation. Although collagen-binding receptors include integrins (α1β1, α2β1, α10β1, and α11β1), all require the β1 subunit. Therefore, using a function-blocking antibody specific for integrin β1 is sufficient to block the interaction of all four integrin heterodimers with collagen. As a result, CD44 blockade did not affect 3D formation, whereas integrin β1 blockade significantly inhibited 3D formation compared to the control group (p < 0.0001), indicating that 3D formation was inhibited probably due to the loose packing of hASCs with CMGs (Fig. 6A and C). These data indicate that hASCs assemble into 3D structures by interacting with CMGs via integrin receptors.

[0155] Integrin activation is known to induce FAK autophosphorylation, which in turn signals to Ras homolog family member A (RhoA) / ROCK, leading to myosin phosphorylation and actin contraction. To confirm that the assembly of 3D structures follows the same signaling pathway, we used FAK (PF-573228), ROCK (Y-27632), and a myosin inhibitor (blebbistatin) to determine whether 3D structure formation was inhibited. As a result, all three inhibitors were found to inhibit 3D structure formation, and this was particularly evident in the 1:4 group, where the size of 3D structures was significantly increased (p < 0.0001). This loose 3D structure formation suggests that integrin activation activates the FAK-ROCK-myosin signaling pathway, leading to actin contraction, which allows hASCs to interact with and compact against CMGs, resulting in the formation of loose 3D structures (Fig. 6 B and C).

[0156] We next hypothesized that increasing the cell:CMG ratio would result in more integrin receptor activation, as more CMGs would be present near the cells. Western blot analysis showed a significant dose-dependent increase in the phosphorylation level of FAK (p < 0.0001) compared to the 1:0 group (Fig. 6D and E).

[0157] Furthermore, since FAK is known to be involved in the suppression of anoikis, we investigated the effects of CD44 and integrin β1 blockade on cell viability. CD44 blockade did not affect cell viability, whereas integrin β1 blockade significantly reduced cell viability for more than 4 days compared to the untreated group (p < 0.0001).

[0158] In summary, when hASCs are cultured with CMG, binding of CMG to integrin receptors on the cell membrane activates the integrin, which phosphorylates FAK. FAK then signals to RhoA and ROCK, which then phosphorylates myosin, leading to actin fiber contraction (Fig. 6F). Blocking integrin β1 or inhibiting FAK, ROCK, or myosin suppresses actin fiber contraction and thus 3D formation. Functionally blocked 3D structures were loosely aggregated due to the inhibition of 3D formation and had lower cell viability than untreated 3D structures.

[0159]

[0160] <Example 4: Confirmation of pores in cell tissue>

[0161] 3D structures assembled with microgels are known to exhibit a porous microstructure. Micro-CT scans were performed to measure the porosity of the 3D structures formed with CMG. As a result, the 1:0 group showed no pores within the structure, which is likely due to the close cell-cell and cell-ECM interactions. Pores were visible starting from the 1:1 group, and the porosity increased with increasing cell:CMG ratio (Figure 7A). Porosity quantification showed a significant increase from near 0% in the 1:0 group to approximately 40% and 60% in the 1:2 and 1:4 ratio groups, respectively (p < 0.0001; Figure 4E). The porous microstructure within the 3D structures enhances mass transport and facilitates the diffusion of nutrients and oxygen into the core. Culturing 3D structures with the glucose analog 2-NBDG revealed that glucose could enter the core via mass diffusion in the 1:1, 1:2, and 1:4 groups, whereas only a limited amount of glucose reached the core of the 1:0 group, indicating limited mass diffusion (Fig. 7B). Under limited mass transfer, cells in the core of 3D structures are reported to experience glucose starvation and hypoxia, which decrease intracellular ATP levels. As a result, intracellular ATP levels significantly increased in a dose-dependent manner with increasing cell:CMG ratios, consistent with the 2-NBDG immunofluorescence data (p < 0.0001) (Fig. 7F).

[0162] Increased mass transfer can facilitate oxygen diffusion into the core of the 3D structure. To investigate the absence of a hypoxic core, the 3D structure was stained with pimonidazole, a hypoxia marker. Immunofluorescence images of cross-sections of the 3D structure showed that the 1:0 group exhibited a hypoxic core, while the 1:1, 1:2, and 1:4 groups showed scattered hypoxic cells throughout the cross-section (Figure 7C).

[0163] To quantify the degree of hypoxia in the 3D structures, Western blotting was performed. As expected, the 1:0 group had the highest degree of hypoxia, and the degree of hypoxia in the other experimental groups decreased with increasing cell:CMG ratio (Fig. 7G). Enhanced mass transfer is expected to increase cell survival. Staining of live and dead cells revealed that more dead cells were observed in the 1:0 group, whereas some dead cells were observed in the 1:1 and 1:2 groups, and nearly all cells in the 1:4 group remained alive at day 7 (Fig. 7D). Consistent with the LIVE / DEAD data, quantification of cell viability revealed that nearly all cells were alive at day 1, while some cells in the 1:0 group began to die by day 3. Overall, a continuous decrease in cell viability was observed through day 28. However, compared to the 1:0 group, a greater number of viable cells were observed in the 1:2 and 1:4 groups (p<0.0001) at all time points (Fig. 7H). To determine the possible causes contributing to the decrease in cell viability with increasing culture time, microCT scans were performed using cells from the 1:4 group to measure changes in porosity over 21 days. The results suggested that the overall size of the 1:4 group continuously decreased with increasing culture time due to cell shrinkage. This was accompanied by a gradual decrease in microporosity within the microstructure, which may ultimately lead to cell death. In summary, increasing the cell:CMG ratio suggests that cells can survive for a longer period of time.

[0164]

[0165] <Example 5: Identification of differentially expressed genes>

[0166] 5-1. To elucidate biological pathways expressed in the 3D structures, mRNA sequencing was performed. DAVID analysis was performed on differentially expressed genes with a >1.5-fold change and p <0.05.

[0167] As a result, we confirmed that biological processes related to actin contraction, mass transfer, anti-apoptosis, and angiogenesis were abundantly expressed in the cell:CMG (1:4 group) sample compared to the cell-only 3D structure (1:0 group) (Fig. 8a).

[0168] Further analysis using heatmaps revealed that genes related to actin contraction, mass transfer, anti-apoptosis, and angiogenesis were upregulated in the cell+CMG 3D construct compared to 2D cultured cells and cell-only 3D (Fig. 8b).

[0169] Analysis of mRNA and protein levels of vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), and TIMP1 (tissue inhibitor matrix metalloproteinase 1), known as an anti-apoptotic factor, which are factors involved in angiogenesis, revealed that the mRNA and protein expression levels of VEGF, IL-8, and TIMP1 increased as the cell:CMG ratio increased (BG in Fig. 8c). In addition, to confirm the effect of a linear and significant decrease in cell viability on the expression levels of VEGF, IL-8, and TIMP1, the secretion of growth factors was quantified, and the protein levels of VEGF, IL-8, and TIMP1 were confirmed to decrease with decreasing cell viability and culture time.

[0170] To evaluate the angiogenic potential in vitro, 3D structures fabricated with cell / CMG ratios of 1:0, 1:1, 1:2, and 1:4 were co-cultured with HUVECs, and the extent of capillary-like tube structure formation induction of the fabricated 3D structures was measured. As the cell:CMG ratio increased, capillary-like tube formation in HUVECs increased in a dose-dependent manner. Specifically, the total length of capillary-like tubes increased by approximately 50% in the 1:0 group and 100% in the 1:4 group (Fig. 9A and B).

[0171] Co-culture of CMG alone with HUVECs did not induce angiogenesis, as evidenced by the low degree of tube formation. This suggests that co-culture of CMG alone in transwells cannot induce angiogenesis.

[0172] To further evaluate the angiogenic capacity of the 3D constructs, 1-mm-thick aortic rings obtained from each group of mice were co-cultured with the 3D constructs. To confirm that the vessels originated from endothelial cells, the aortic rings were fixed and stained with lectin-IB4, an endothelial cell marker. As a result, it was found that culturing aortic rings with EBM or CMG resulted in almost no microvessel formation due to the lack of angiogenic factors. Increasing the cell:CMG ratio from 1:0 to 1:4 significantly increased the degree of microvessel formation (approximately 10% to 70%, respectively), and the 1:4 group showed the highest microvessel formation (Fig. 9C and D).

[0173] In vitro and ex vivo experimental results suggest that 3D constructs with increased cell:CMG ratios exhibit enhanced angiogenic potential. This suggests that increased cell:CMG ratios lead to increased secretion of angiogenic factors, resulting in HUVEC sprouting from the aortic ring and microangiogenesis.

[0174]

[0175] <Example 6: Administration of 3D cell tissue to a model of severe hypolipidemia>

[0176] In animal experiments, a model of severe hypolipidemia induced by transplanting the paste-type 3D cell tissue of the present invention demonstrated restoration of blood flow and reduction in necrosis. The 3D cell tissue used in the experiment was selected from a 1:4 group with high angiogenic potential in vitro and ex vivo. The stem cell complex formulation of the present invention, which injected 200,000 cells, showed significantly superior results compared to previous animal experiments that injected approximately 2 million cells.

[0177] Specifically, after inducing ischemia, PBS, CMG alone, CMG / hASCs suspension, cell-only 3D construct (1:0 group), or 3D construct (1:4 group) were injected into the ischemic limb on Day 1, and the morphology and blood flow were observed using laser Doppler imaging (Fig. 10, A). On Day 28, the blood flow in the group injected with the CMG / hASCs suspension increased by approximately 40% compared to the contralateral limb (Fig. 10, B), and 20% of the ischemic limb was rescued (Fig. 10, C), whereas the 3D construct (1:0) and CMG injection groups showed no significant changes compared to the PBS injection group. However, the 3D construct (1:4) injection group showed 20% higher blood flow compared to the CMG / hASCs suspension injection group (Fig. 7, B), and 60% of the ischemic limb was recovered (Fig. 10, C).

[0178] To investigate whether the degradation rate of CMG in the CMG-hASC 3D constructs was related to the therapeutic efficacy, an in vitro enzymatic biodegradation assay was performed, and the biodegradation rate was tracked using an in vivo imaging system (IVIS). In vitro, the degradation rates of the 1:1, 1:2, and 1:4 3D constructs were much lower than those of CMG alone. This is thought to be due to easier access of collagenase in the absence of cells. In vivo, the biodegradation rate was analyzed in ischemic and healthy limbs using Atto-488-conjugated CMG. The biodegradation rate of CMG did not differ from that of the collagen / HA gel, suggesting that the degree of biodegradation through gel fragmentation was not different. The fluorescence signal of the CMG / hASC 3D construct decreased more slowly than that of the collagen / HA gel, CMG alone, and CMG / hASC suspension groups, suggesting that the degradation rate of CMG in the 3D constructs was slow. However, in all groups, the fluorescent signal gradually decreased at a similar rate over 3 weeks and completely disappeared 4 weeks after injection into the ischemic limb.

[0179] In summary, the 3D CMG / hASC construct, assembled with cells, exhibits slow biodegradation, suggesting that CMG can support cells at the transplant site in vivo, potentially impacting therapeutic efficacy. Meanwhile, low biodegradation rates were not observed in ischemic hindlimbs, suggesting that degradation is accelerated by a robust immune response following ischemia.

[0180]

[0181] <Example 7. Selection of the optimal size of a 3D cell tissue>

[0182] Additionally, to determine the effect of the size of the 3D structure of CMG / hASC on cell viability and therapeutic efficacy, CMG-hASC was prepared at a volume ratio of 1:4, but the cells were 5 x 10 4, 10 x 10 4 , 20 x 10 4 , or 40 x 10 4 3D structures with a ratio of 1:4 were fabricated by varying the size of the CMG / hASC 3D structures. The diameters of the fabricated 3D structures were confirmed to be 2 mm, 2.5 mm, 3 mm, or 4.2 mm, respectively. The results indicate that as the size of the 3D structure of CMG / hASC increases, the secretion of VEGF, IL-8, and TIMP1 increases, but the cell viability decreases. As a result of injection of 3D structures of CMG / hASC of various sizes, 10 x 10 4 or 20 x 10 4 3D constructs of 2.5 mm or 3 mm in size of the canine cells significantly increased blood perfusion at day 7 compared to the PBS-treated group, suggesting that the size of the 3D construct showing optimal therapeutic efficacy is 2.5 mm or 3 mm.

[0183]

[0184] <Example 8: Mechanism Verification in a Model of Severe Hyperlipidemia Induced by Administration of 3D Cell Tissue>

[0185] In vivo, cell viability was confirmed by human nuclear antigen (HNA) staining in ischemic muscles. HNA-positive cells were not evident in the contralateral muscle, PBS, or CMG alone groups. HNA-positive cells were observed for more than 28 days in the CMG / hASC 3D construct group, suggesting that transplanted cells survived until day 28. However, in the CMG / hASC suspension group and the cell aggregates alone group, HNA-positive cells were observed only until day 14 after administration (Fig. 11a). In the CMG / hASC 3D construct group, HNA-positive cells were significantly higher up to day 7 (p < 0.01) and day 14 (p < 0.05) compared to the CMG / hASC suspension and cell aggregates alone groups (Fig. 11d). In summary, the 3D structure of CMG / hASCs showed superior cell delivery effect than CMG / hASCs suspension or cell aggregates and ensured long-term cell survival after injection.

[0186] The muscle regenerative potential of the 3D CMG / hASC construct was assessed by staining cross-sections of thigh muscles with laminin and counting the number of regenerated muscle fibers with concentrated nuclei. At day 14, a small number of regenerated muscle fibers were observed in the CMG / hASC suspension and cell aggregate administration groups, whereas a significantly higher number of regenerated muscle fibers was observed in the CMG / hASC 3D construct administration group, suggesting that extensive muscle fiber regeneration occurred at day 14 (p < 0.0001).

[0187] At day 28 after administration, more centrally located nuclei were observed in the CMG / hASCs suspension and cell-only aggregate groups, indicating that some muscle regeneration occurred at day 28 (p < 0.05). However, the CMG-hASCs 3D construct group showed a significantly reduced number of centralized nuclei compared to the CMG / hASCs suspension and cell-only aggregate groups at day 28, and showed recovery to a muscle structure similar to that of the contralateral leg, demonstrating the highest muscle regeneration potential (Figs. 11b and 11e).

[0188] To evaluate the angiogenic potential of the 3D constructs of CMG / hASCs, muscle tissues were stained with CD31, a marker of endothelial cells. CD31-positive cells were observed in all treatment groups from day 14 onward, and the 3D construct group of CMG / hASCs had a significantly higher number of CD31-positive cells than the CMG / hASCs suspension and cell aggregate groups (Figs. 11c and 11f). On day 28, more CD31-positive cells were observed in all treatment groups than on day 14. However, the 3D construct group of CMG / hASCs had a significantly higher number of CD31-positive cells than the CMG / hASCs suspension and cell aggregate groups (p < 0.0001). In contrast, low levels of CD31-positive cells were observed in the PBS and CMG alone treatment groups on both days 14 and 28.

[0189]

[0190] <Example 9: Confirmation of particle size according to hyaluronic acid concentration>

[0191] 7-1. As the HA concentration increased, the size of the microgel fragments tended to decrease, while as the collagen concentration increased, the size of the microgel fragments tended to increase (A and B in Fig. 12).

[0192] 7-2. The most frequent size of the microgel fragments manufactured using 10% collagen and 5% HA was 30 um in length and 20 um in width (C and D in Fig. 12).

[0193]

[0194] <Example 10: Effect of hyaluronic acid concentration in 3D cell structures>

[0195] 8-1. As the HA concentration increased from 0% to 5%, the diameter of the 3D cell structure decreased (A and B in Fig. 13).

[0196] 8-2. The storage and loss modulus was greatest at 0% HA and 10% collagen concentrations, and showed a tendency to decrease as the HA concentration increased (C in Figure 13).

[0197] 8-3. 3D structures formed using collagen + 0% HA and 0.5% HA had microgel fragments remaining, whereas 3D structures formed using collagen + 1%-5% produced a single 3D structure with no microgel fragments remaining (Fig. 13D).

[0198]

[0199] <Example 11: Confirmation of microgels showing uniform cell distribution>

[0200] 2x10 5 Cross-sections of 3D cell structures formed by culturing hASCs in collagen microgels at a 1:4 ratio were subjected to immunofluorescence staining. The cell-to-CMG ratio was determined by pellet volume. Microgels were prepared by mixing 10% collagen with various concentrations of hyaluronic acid ranging from 0% to 5%.

[0201] Increasing the hyaluronic acid concentration resulted in the formation of 3D structures with a more uniform cell distribution.

[0202]

[0203] <Example 12: Effect of collagen concentration on 3D cell structures>

[0204] 10-1. The diameter of the 1:4 cell:CMG cell tissue increased with increasing collagen concentration.

[0205] 10-2. The storage and loss modulus of the three-dimensional cell tissue increased with increasing collagen concentration.

[0206] 10-3. The three-dimensional structures formed with 12.5% ​​hyaluronic acid and 15% collagen left microgel fragments, but the three-dimensional structures with 5 to 10% collagen content did not leave microgel fragments.

[0207]

[0208] <Example 13: Effect of collagen concentration on 3D cell structures>

[0209] 2x10 5 Cross-sections of three-dimensional cell structures formed by culturing hASCs in collagen microgels at a 1:4 ratio were subjected to immunofluorescence staining. The cell-to-CMG ratio was determined by pellet volume. Microgels were prepared by mixing 2.5% hyaluronic acid with various collagens ranging from 5% to 15%.

[0210] Increasing the collagen concentration hindered the formation of 3D structures with a more uniform distribution of cells.

[0211]

[0212] Although the embodiments described above have been described with limited drawings, those skilled in the art will appreciate that various technical modifications and variations can be applied based on the above. For example, appropriate results can still be achieved even if the described techniques are performed in a different order than described, and / or components of the described systems, structures, devices, circuits, etc. are combined or combined in a different manner than described, or are replaced or substituted with other components or equivalents.

[0213] Therefore, other implementations, other embodiments, and equivalents to the claims also fall within the scope of the claims described below.

Claims

A microgel composition comprising 9.5% to 10.5% (w / v) collagen and 4.5 to 5.5% (w / v) hyaluronic acid.

2. In paragraph 1, A microgel composition, wherein the average size of the microgel is 20 to 50 μm. A three-dimensional scaffold comprising 9.5% to 10.5% (w / v) collagen and 4.5% to 5.5% (w / v) hyaluronic acid.

4. In paragraph 3, The above three-dimensional scaffold is a three-dimensional scaffold loaded with cells. A three-dimensional cell structure produced by co-culturing cells with a microgel containing 9.5% to 10.5 (w / v) collagen and 4.5 to 5.5% (w / v) hyaluronic acid.

6. In paragraph 5, The above three-dimensional cell structure is a three-dimensional cell structure produced by mixing microgel and cells in a volume ratio of 1:3 to 5.

7. In paragraph 5, A three-dimensional cell structure wherein the above cells are stem cells.

8. In paragraph 7, A three-dimensional cell structure wherein the above stem cells are adipose-derived stem cells.

9. A pharmaceutical composition for treating peripheral arterial disease comprising a three-dimensional cell structure as an active ingredient, A pharmaceutical composition wherein the three-dimensional cell structure is produced by culturing cells together with a microgel containing 9.5% to 10.5 (w / v) collagen and 4.5 to 5.5% (w / v) hyaluronic acid.

10. In paragraph 9, A pharmaceutical composition wherein the above three-dimensional cell structure is produced by mixing and culturing the microgel and cells in a volume ratio of 1:3 to 5.

11. In paragraph 9, A pharmaceutical composition, wherein the above pharmaceutical composition is in the form of an injection.

12. In paragraph 9, A pharmaceutical composition wherein the above cells are adipose-derived stem cells.

13. In paragraph 9, A pharmaceutical composition wherein the peripheral arterial disease is severe limb ischemia. 14.(1) A step of mixing 9.5% to 10.5 (w / v) collagen and 4.5 to 5.5% (w / v) hyaluronic acid and incubating to gel; (2) a step of preparing a microgel by stirring the mixture after gelation; and (3) A method for manufacturing a three-dimensional cell structure, comprising a step of mixing and culturing the microgel and cells.

15. In paragraph 14, The above step (3) is a method for manufacturing a three-dimensional cell structure, wherein the microgel and cells are mixed and cultured in a volume ratio of 1:3 to 5.

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