Stem cell isolation kit manufacturing composition comprising graphene, and preparation method therefor

Abstract

The present invention relates to: a stem cell isolation kit manufacturing composition prepared with graphene dispersed therein, and a preparation method therefor, the composition enabling cell activity of stem cells to improve and blood clots to be prevented through mixing of graphene and a thermoplastic resin. The stem cell isolation kit manufactured using the stem cell isolation kit manufacturing composition comprising graphene, of the present invention, allows autologous stem cells to be activated by an environment in which biocompatible far-infrared (FIR) emission and electrical conductivity are increased, and, under hypoxic conditions, enables stem cell water amount to be increased through low oxygen permeability (water activity). In addition, the present invention is subjected to antibacterial treatment so as to provide an antibacterial effect, and can improve a platelet adsorption reduction rate through a water slip effect of the graphene composition.

Description

Composition for manufacturing a stem cell isolation kit containing graphene and method for manufacturing the same

[0001] This application claims priority to Korean Patent Application No. 10-2024-0172107 filed on November 27, 2024, and the entire specification is a reference to this application.

[0002]

[0003] The present invention relates to a composition for manufacturing a stem cell isolation kit in which graphene is dispersed, which can improve cell activity and prevent blood clots by creating an environment with increased electrical conductivity through mixing graphene and a thermoplastic resin, and a method for manufacturing the same.

[0004]

[0005] Graphene is regarded as the "dream nanomaterial" and "the material with the most outstanding properties among existing materials." Graphene is obtained by peeling off the thinnest single layer from graphite, which consists of a structure of stacked hexagonal honeycomb layers. With a thickness of 0.2 nm, it is highly transparent and can transmit 100 times more current than copper and 100 times faster than silicon. Its tensile strength is 130 GPa, which is more than 200 times stronger than the tensile strength of steel (less than 2 GPa), and due to its excellent elasticity, it does not lose electrical conductivity even when deformed. Furthermore, graphene's thermal conductivity ranges from 4,800 to 5,300 W / mk, which is more than twice as high as that of diamond, known for having the highest thermal conductivity of 900 to 2,300 W / mk.

[0006] Stem cells refer to a broad concept encompassing undifferentiated cells possessing "stemness"—that is, the ability to differentiate into various types of body tissue cells. These stem cells are broadly classified into embryonic stem cells (which can be produced using embryos), adult stem cells, gametes, and cancer stem cells. Because these stem cells are capable of differentiating into diverse cell types and can be utilized in various ways, such as suppressing excessive immune responses, acting as vectors for gene therapy, and producing various growth factors, they are being actively researched as novel cell therapies.

[0007] Recently, research has been conducted to apply the properties of graphene to stem cell culture or differentiation. However, when stem cells are cultured or differentiated using graphene-coated culture equipment, there is a possibility that graphene may be internalized into the stem cells, which could lead to toxicity.

[0008] On the other hand, no research has been conducted on enhancing the physiological activity of stem cells by mixing graphene with thermoplastic resin and applying this mixture to the manufacture of a kit capable of isolating stem cells, rather than by coating with graphene.

[0009]

[0010] [Prior Art Literature]

[0011] [Patent Literature]

[0012] (Patent Document 1) KR 10-2018-0041267 (2018-04-09)

[0013]

[0014] As a result of diligent efforts to produce a stem cell isolation kit that can apply the advantages of graphene to stem cells without causing toxicity to them, the inventors confirmed that when a stem cell isolation kit is manufactured by compounding graphene with a thermoplastic resin at an optimal concentration without coating, the physiological activity of the stem cells is enhanced by an environment with increased electrical conductivity without causing toxicity to the stem cells, and thus completed the present invention.

[0015] Accordingly, the objective of the present invention is to provide a composition for manufacturing a stem cell isolation kit in which graphene is dispersed by mixing graphene and a thermoplastic resin, and a method for manufacturing the same.

[0016]

[0017] The present invention provides a composition for manufacturing a stem cell isolation kit comprising graphene and a thermoplastic resin, wherein the composition is characterized by improving cell activity of stem cells and preventing blood clots through an environment in which electrical conductivity is increased.

[0018] According to a preferred embodiment of the present invention, the graphene is added as a powder.

[0019] According to a preferred embodiment of the present invention, the graphene is added at a concentration of 0.01-0.04 wt%.

[0020] According to a preferred embodiment of the present invention, the thermoplastic resin is one or more selected from the group consisting of PET (Poly Ethylene Terephthalate), PA (Polyamide), PC (Polycarbonate), PE (Polyethylene), PP (Polypropylene), PLA (Polylactic acid) and PBAT (Polybutylene Adipate-co-Terephthalate).

[0021] According to a preferred embodiment of the present invention, the stem cell is an adult stem cell.

[0022] The present invention also comprises: i) a step of preparing a mixture by mixing graphene and a thermoplastic resin;

[0023] ii) a step of manufacturing graphene composite material pellets by injection and extrusion of the above mixture; and

[0024] iii) A step of forming the above pellets;

[0025] A method for manufacturing a stem cell isolation kit comprising,

[0026] The kit manufactured by the above manufacturing method provides a manufacturing method characterized by improving cell activity of stem cells and preventing blood clots through an environment with increased electrical conductivity.

[0027] The present invention also provides a stem cell isolation kit manufactured by the above manufacturing method.

[0028]

[0029] The present invention will be described in more detail below.

[0030]

[0031] The term “water activity” in the present invention refers to a method of expressing water content that takes into account relative humidity and the water actually available to microorganisms, instead of expressing the water content of a substance as a %, and may mean the ratio of the water vapor pressure (Ps) of a substance at any temperature to the water vapor pressure (Po) of pure water at that temperature.

[0032] The term “water slip” in the present invention may refer to the sliding of water, and may refer to a surface that is smooth so that water or other substances such as cells slide off without adhering to the surface.

[0033]

[0034] The inventors of the present invention have completed the present invention to produce a composition for manufacturing a stem cell isolation kit that can apply the advantages of graphene to stem cells without causing toxicity to the stem cells.

[0035] The present invention utilizes a composition for manufacturing a stem cell isolation kit containing 0.01-0.04 wt% of liquid graphene, thereby exhibiting unique color and transparency in the kit (Fig. 1) and enhancing the physiological activity of stem cells isolated from the kit. Specifically, the stem cell isolation kit prepared with the composition containing graphene according to the present invention—that is, in which graphene is dispersed—enables autologous stem cells to be activated by an environment with increased biocompatible far-infrared (FIR) emission and electrical conductivity, thereby improving cell activity. Additionally, the oxygen permeability (water activity) is low under hypoxic conditions, which can improve the stem cell water content. Furthermore, the kit is treated with an antibacterial agent to provide an antibacterial effect, and the reduction rate of platelet adsorption due to the water slip effect of the graphene composition can prevent blood clots.

[0036]

[0037] Accordingly, the present invention can provide a composition for manufacturing a stem cell isolation kit comprising graphene and a thermoplastic resin, wherein the composition is characterized by improving cell activity of stem cells and preventing blood clots through an environment in which electrical conductivity is increased.

[0038] According to a preferred embodiment of the present invention, the graphene may be added as a powder. Adding it as a powder may be preferable for the dispersion of graphene within the kit.

[0039] According to a preferred embodiment of the present invention, the graphene may be added at a concentration of 0.01-0.04 wt%. Preferably, the graphene may be added at a concentration of 0.01 wt%.

[0040] The transparency of the stem cell isolation kit of the present invention can be controlled by the amount of graphene added. If graphene is added in an amount less than 0.01 wt%, there are problems with the desired far-infrared and oxygen permeability results, and if it is added in an amount exceeding 0.04 wt%, transparency may be lost.

[0041] According to a preferred embodiment of the present invention, the thermoplastic resin may be one or more selected from the group consisting of PET (Poly Ethylene Terephthalate), PA (Polyamide), PC (Polycarbonate), PE (Polyethylene), PP (Polypropylene), PLA (Polylactic acid) and PBAT (Polybutylene Adipate-co-Terephthalate).

[0042] According to a preferred embodiment of the present invention, the stem cell may be an adult stem cell. The adult stem cell may be an adult stem cell derived from blood, bone marrow, or fat, preferably an adult stem cell derived from blood, and more preferably a blood-derived CD34+ adult stem cell.

[0043] The stem cell isolation kit of the present invention may include, but is not limited to, a platelet-rich plasma (PRP) chamber cap, a PRP chamber, a PRP locking bar, a plasma chamber, a red blood cell (RBC) locking bar, an RBC chamber cap, and an RBC chamber (Fig. 2).

[0044]

[0045] The present invention also comprises: i) a step of preparing a mixture by mixing graphene and a thermoplastic resin;

[0046] ii) a step of manufacturing graphene composite material pellets by injection and extrusion of the above mixture; and

[0047] iii) A step of forming the above pellets;

[0048] A method for manufacturing a stem cell isolation kit comprising,

[0049] A kit manufactured by the above manufacturing method can provide a manufacturing method characterized by improving cell activity of stem cells and preventing blood clots through an environment with increased electrical conductivity.

[0050] According to a preferred embodiment of the present invention, the graphene may be added as a powder. Adding it as a powder may be preferable for the dispersion of graphene within the kit.

[0051] According to a preferred embodiment of the present invention, the graphene may be added at a concentration of 0.01-0.04 wt%. Preferably, the graphene may be added at a concentration of 0.01 wt%.

[0052] The transparency of the stem cell isolation kit of the present invention can be controlled by the amount of graphene added. If graphene is added in an amount less than 0.01 wt%, there are problems with the desired far-infrared and oxygen permeability results, and if it is added in an amount exceeding 0.04 wt%, transparency may be lost.

[0053] According to a preferred embodiment of the present invention, the thermoplastic resin may be one or more selected from the group consisting of PET (Poly Ethylene Terephthalate), PA (Polyamide), PC (Polycarbonate), PE (Polyethylene), PP (Polypropylene), PLA (Polylactic acid) and PBAT (Polybutylene Adipate-co-Terephthalate).

[0054] Graphene composite pellets are intended to simplify the handling of thermoplastic resins, and their size may be approximately 0.1 mm to 100 mm, and they may be in the form of spherical, cylindrical, or prismatic granules.

[0055] According to a preferred embodiment of the present invention, the stem cell may be an adult stem cell. The adult stem cell may be an adult stem cell derived from blood, bone marrow, or fat, preferably an adult stem cell derived from blood, and more preferably a blood-derived CD34+ adult stem cell.

[0056] The stem cell isolation kit of the present invention may include, but is not limited to, a platelet-rich plasma (PRP) chamber cap, a PRP chamber, a PRP locking bar, a plasma chamber, a red blood cell (RBC) locking bar, an RBC chamber cap, and an RBC chamber (Fig. 2).

[0057]

[0058] The present invention may also provide a stem cell isolation kit manufactured by the above manufacturing method.

[0059]

[0060] A stem cell isolation kit prepared using the composition for manufacturing a stem cell isolation kit containing graphene according to the present invention can activate autologous stem cells through an environment with increased biocompatible far-infrared (FIR) emission and electrical conductivity, and can improve the stem cell water content (Water Amount) by having a low oxygen permeability (water activation) in a hypoxic state (Hypoxia). In addition, it is treated with an antibacterial agent to provide an antibacterial effect, and can improve the rate of reduction in platelet adsorption due to the water slip effect of the graphene composition.

[0061]

[0062] Figure 1 shows the stem cell isolation kit of the present invention manufactured including graphene (left) and the stem cell isolation kit manufactured without graphene (right).

[0063] Figure 2 shows the structure of the stem cell isolation kit of the present invention.

[0064] FIG. 3a shows the external appearance of the stem cell isolation kit of the present invention manufactured including graphene (lid attached state).

[0065] FIG. 3b shows the external appearance of the stem cell isolation kit of the present invention manufactured with graphene (lid separated).

[0066] Figure 4 shows the stem cell isolation kit of the present invention, which exhibits a difference in transparency depending on the graphene content (A: 0.03wt%, B: 0.01wt%).

[0067] Figure 5 shows scanning electron microscopy (SEM) images of a stem cell isolation kit manufactured without graphene and a stem cell isolation kit of the present invention manufactured with graphene.

[0068] Figure 6 shows the results of confirming cell viability for the stem cell isolation kit of the present invention manufactured including graphene.

[0069] Figure 7 shows the results of confirming the cell culture form for the stem cell isolation kit of the present invention manufactured including graphene.

[0070] FIG. 8 shows a TEM (Transmission Electron Microscopy) image of the stem cell isolation kit of the present invention manufactured including graphene (resolution: 2048 × 2048 pixels).

[0071]

[0072] The present invention will be explained in more detail below through examples. These examples are intended solely to illustrate the present invention, and it is obvious to those skilled in the art that the scope of the present invention is not to be interpreted as being limited by these examples.

[0073]

[0074] [Example 1]

[0075] Manufacturing of graphene-dispersed stem cell isolation kits

[0076] Medical-grade polycarbonate (PC) powder and micro-sized graphene powder (Haydale HDPlas® GNP / FLG, Table 1) were blended as a thermoplastic resin and dispersed without a dispersion agent. The amount of graphene added relative to the thermoplastic resin was set to 0.01 wt%. The mixed resin and graphene were injection-molded and extruded using a molding machine or a compression roller to produce graphene composite pellets mixed with thermoplastic resin and graphene. Next, the graphene composite pellets were molded using a molding machine to form a finished stem cell isolation kit.

[0077] Data Measurement Method Bulk Density ~ 215 kg / m³ 3 EN ISO 60 Amorphous Carbon Undetected SEM / TEM Specific Surface Area ~ 20 m 2 / gBET Analysis GNP Planar Size 0.3 ~ 5 μm SE GNP Thickness < 50 nm SEM

[0078]

[0079] [Example 2]

[0080] Measurement of Far-infrared Emissivity and Antibacterial Activity of Graphene-Dispersed Stem Cell Isolation Kit

[0081] <2-1> Far-infrared emissivity

[0082] Far Infrared Radiation (FIR) has a wavelength of 4 to 16 microns and can help promote health by promoting blood circulation, removing toxins, and strengthening immunity. We intended to determine the far infrared emissivity of the stem cell isolation kit containing dispersed graphene (PCA and PCB) of the present invention prepared in <Example 1> above.

[0083] The Korea Institute of Far Infrared Application Evaluation (KIFA) was commissioned to measure the emissivity using an FT-IR spectrometer at 37°C in comparison with a black body. The measurement standard was KFIA-FI-1005.

[0084] Far-infrared emissivity (5 ~ 20 µm) Graphene PCA 0.893 Graphene PCB 0.89

[0085] As a result, as shown in [Table 2] above, it was confirmed that the stem cell isolation kit of the present invention emits biocompatible far-infrared rays.

[0086]

[0087] <2-2> Antibacterial power

[0088] We intended to measure the antibacterial activity of the stem cell isolation kit containing dispersed graphene (PCA and PCB) of the present invention prepared in <Example 1> above.

[0089] The test was commissioned to the Korea Institute of Far Infrared Application Evaluation (KIFA), and the measurement standard was KFIA-FI-1003. The bacterial strains used in the antibacterial activity test were Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 6538). Blank refers to the measurement taken without adding a sample, and the number of bacteria on the culture medium was calculated by multiplying by the dilution factor.

[0090] Strain Sample Initial Concentration (CFU / ml) Concentration after 24 hours (CFU / ml) Bacterial Decrease Rate (%) E. coli Blank 2.2×10 5 7.5×10 6 - Graphene PCA 1.1×10 5 98.5 Staphylococcus Blank 2.0×10 5 6.9×10 6 - Graphene PCA 7.0×10 4 99.0

[0091] Strain Sample Initial Concentration (CFU / ml) Concentration after 24 hours (CFU / ml) Bacterial Decrease Rate (%) E. coli Blank 2.1×105 7.3×10 6 - Graphene PCB 2.8×10 5 96.2 Staphylococcus Blank 2.4×10 5 6.6×10 6 - Graphene PCB 1.2 × 10⁻⁶ 5 98.2

[0092] As a result, as shown in [Table 3] and [Table 4] above, it was confirmed that the stem cell isolation kit of the present invention exhibits excellent antibacterial activity.

[0093]

[0094] [Example 3]

[0095] Water Slip Effect

[0096] Scanning electron microscopy (SEM) images were examined to confirm the water slip effect of the graphene-dispersed stem cell isolation kit of the present invention prepared in <Example 1> above.

[0097] As a result, as shown in [Fig. 5], in the case of a stem cell isolation kit manufactured without graphene, the surface is not smooth, so adsorption of platelets, etc. may occur, whereas in the case of a stem cell isolation kit manufactured with graphene of the present invention, the surface is smooth, and it was confirmed that adsorption of platelets, etc. is significantly reduced due to the water slip effect.

[0098]

[0099] [Example 4]

[0100] Toxicity test

[0101] We intended to verify the cytotoxicity of the graphene-dispersed stem cell isolation kit of the present invention prepared in <Example 1> above.

[0102] Specifically, NIH 3T3 and MRC-5 cells were obtained from the Korean Cell Line Bank (Seoul, Korea) and cultured. DMEM was used as the cell culture medium, and the cells were cultured at 37°C in a 5% CO2 incubator. Cells (1 x 10⁶ 4 After cutting and adding the above kit (samples; PCA, PCB) to wells in which cells were cultured, the cells were incubated for 24, 48, and 72 hours, and cytotoxicity was measured using the MTT assay method. The absorbance of the MTT samples at 570 nm was measured using a microplate reader. Cytotoxicity was evaluated based on ISO 10993-5:2009, where cell viability decreased by 30% or more.

[0103] Additionally, NIH 3T3 and MRC-5 cells were cultured in a cell culture plate, and the above kit was added. After co-culturing the cells with GRP for 24, 48, and 72 hours, the proliferation and morphology of the cells were observed using an optical microscope.

[0104] As a result, as shown in [Fig. 6] and [Fig. 7], it was confirmed that there was no change in cell viability in the kit of the present invention, and no specific morphology was observed in the cultured cells, thus confirming that there was no cytotoxicity.

[0105]

[0106] [Example 5]

[0107] Verification of Graphene Dispersion Characteristics_TEM Analysis

[0108] Transmission Electron Microscopy (TEM) analysis was performed to confirm the dispersion state of nano-sized graphene powder present in the polycarbonate (PC) of the graphene composite material pellets prepared in <Example 1> above. The prepared sample was prepared to a thickness that allows the electron beam to penetrate, mounted on a TEM grid, and then transmission electron microscopy analysis was performed under a set total magnification of approximately x286,400 (display magnification x12,000) and an acceleration voltage (300 kV) to confirm its morphological characteristics.

[0109] As a result, as shown in [Figure 8], it was confirmed that the graphene powder maintained a layered structure and was uniformly dispersed within the polycarbonate (PC) resin, minimizing aggregation.

[0110]

[0111] A stem cell isolation kit manufactured using the composition for manufacturing a stem cell isolation kit containing graphene according to the present invention can activate autologous stem cells through an environment with increased biocompatible far-infrared (FIR) emission and electrical conductivity, and can improve the stem cell water content (Water Amount) by having a low oxygen permeability (water activation rate) in a hypoxic state (Hypoxia). In addition, it has an antibacterial effect due to antibacterial treatment and can improve the rate of reduction in platelet adsorption due to the water slip effect of the graphene composition, thus having industrial applicability.

Claims

1. A composition for manufacturing a stem cell isolation kit comprising graphene and a thermoplastic resin, wherein The above composition is characterized by improving cell activity of stem cells and preventing blood clots through an environment of increased electrical conductivity.

2. A composition according to claim 1, characterized in that the graphene is added in powder form.

3. A composition according to claim 1, characterized in that the graphene is added at a concentration of 0.01-0.04 wt%.

4. A composition according to claim 1, wherein the thermoplastic resin is one or more selected from the group consisting of PET (Poly Ethylene Terephthalate), PA (Polyamide), PC (Polycarbonate), PE (Polyethylene), PP (Polypropylene), PLA (Polylactic acid), and PBAT (Polybutylene Adipate-co-Terephthalate).

5. A composition according to claim 1, characterized in that the stem cells are adult stem cells. 6.i) A step of preparing a mixture by mixing graphene and a thermoplastic resin; ii) a step of manufacturing graphene composite material pellets by injection and extrusion of the above mixture; and iii) A step of forming the above pellets; A method for manufacturing a stem cell isolation kit comprising, A manufacturing method characterized by the kit manufactured by the above method improving cell activity and preventing blood clots through an environment with increased electrical conductivity.

7. A stem cell isolation kit manufactured by the manufacturing method of Paragraph 6.

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