KR20160016687A - Transplantable graphene-nanofibrous scaffold applied to brain disease, and photoacoustic imaging for tracking the same - Google Patents

Abstract

The present invention relates to a brain disease application technology using a graphene-nanofiber support for transplant and a photo-acoustic image technology for tracking a graphene-nanofiber support in vivo. A support for culturing cells comprises: graphene; and nanofibers coming into contact with the graphene.

Description

TECHNICAL FIELD [0001] The present invention relates to graphene-nanofiber scaffold for transplantation, and to photonic-acoustical imaging technology for tracking the graphene nanofiber scaffold. [0002]

The present invention relates to a brain injury model application technique using a grafting-nanofiber support for implantation, and a photoacoustic imaging technique for biopsy of the graphene-nanofiber support.

Low-dimensional nanomaterials composed of carbon atoms include fullerene, carbon nanotube, graphene, and graphite. That is, they are divided into fullerenes having a hexagonal arrangement of carbon atoms, spherical carbon nanotubes, graphene arranged as one layer of carbon atoms, and graphite stacked in three dimensions. In particular, graphene moves electrons about 100 times faster than silicon, theoretically, it can flow about 100 times more current than copper, and it is a new material that has been studied a lot recently due to its high strength and elasticity and light weight.

The support obtained from plant and animal resources is biocompatible and biodegradable as well as hydrophilic because of its adherence to the hydrophobic synthetic support as compared to the hydrophobic support. High, but mechanical properties are lower than synthetic supports.

In recent years, therapeutic methods for expressing brain forming proteins or promoting the differentiation of stem cells into brain cells have been studied. Particularly, studies on artificial brain having a 3D cell culture structure including synthetic molecules and biomolecules that cause biochemical mechanisms and enabling effective brain cell regeneration and proliferation are underway. Therefore, a support component which is harmless to the human body is required. At this time, a substance such as carbon nanotubes and graphene composed of carbon atoms can be expected as such a support component.

Stem cells are the cells that constitute biological tissues. They are cells that can differentiate into various cells. They are the cells that can be obtained from different tissues of the embryo, fetus, and adult, Undifferentiated cells are collectively called. Unlike the differentiated cells in which the cell division is stopped, the stem cells are characterized by being capable of self-renewal to the same cells as themselves by cell division. In addition, when the differentiation stimulus is given by the culture environment, And a stem cell can differentiate into various types of cells.

Particularly, it is the cell therapy agent using such stem cells that has been most interested in the treatment of various diseases. On the other hand, in order for the transplanted stem cells to be effective in the treatment of diseases, at least two requirements must be met. The first requirement is that the transplanted stem cells differentiate into the desired cells. The second requirement is that the properly differentiated stem cells for therapy are networked with the transplanted tissue to replace the damaged tissue in the transplanted tissue and perform its function well.

However, current stem cell differentiation techniques have difficulty in genetically differentiating stem cells into desired cells. In order to solve this problem, it is important to find extracellular factors affecting the differentiation of stem cells into specific cells. Most of the signals related thereto are not yet known. However, platelet growth factor (PDGF), epithelial growth factor ), And retinoic acid have been found to induce differentiation into specific cells. In addition, direct stem cell transplantation to the injured site is washed there without being networked by a number of factors. Accordingly, there have been studies on the use of various substances as supports in the culture of stem cells. For example, there have been related studies such as "Method for modifying the surface of a polymer scaffold for stem cell transplantation using a depleted extracellular matrix [Korean Published Patent Application No. 2010-0114815] ".

However, in order to use stem cells for the treatment of damage of brain, cartilage, etc., it is further required that high purity of desired cells and ease of clinical application are required. In other words, a biocompatible cell structure support is needed that can promote the differentiation of stem cells into specific cells, while assisting in the formation of a network with a living body in vivo application. Recently, there has been a study of scaffolds for three-dimensional culture of stem cells. Such scaffolds have been shown to increase pore size, porosity, and interconnectivity of pores, allowing cells to easily penetrate into the scaffold And should be characterized by facilitating nutrient circulation and oxygen supply.

Accordingly, equipment using solid freeform fabrication has been developed. Typical examples thereof include stereolithography (SLA), selective laser sintering (SLS), fused deposition modeling (FDM), 3D printing ) Method, and 3D plotting method. However, there is a growing demand for the development of scaffolds that enhance the therapeutic effect of stem cell - mediated therapeutic agents beyond the simple cultivation of stem cells and enhance the differentiation efficiency of stem cells into specific cells, thereby facilitating their application in clinical practice.

The present invention provides a brain injury model application technique using a grafting-nanofiber support for grafting and a photoacoustic imaging technique for biopsy of the graft-nanofiber support.

However, the problems to be solved by the present invention are not limited to the above-mentioned problems, and other problems not mentioned can be clearly understood by those skilled in the art from the following description.

A first aspect of the present invention provides a support for cell culture comprising graphene and nanofibers bonded with the graphene.

A second aspect of the present invention provides a method for producing stem cells comprising culturing stem cells on a cell culture support comprising a graphene-nanofiber support comprising a nanofiber support in combination with graphene according to the first aspect of the present invention, .

A third aspect of the present invention provides a stem cell cultured on a graphene-nanofiber support by the method of cultivation of the second aspect of the present application.

A fourth aspect of the invention is a cell therapy composition for treating brain diseases comprising cells supported on a graphene-nanofiber support comprising a nanofiber support coupled with graphene according to the first aspect of the present application .

According to a fifth aspect of the invention, there is provided a method of treating cancer comprising administering to a animal a composition for cell therapy comprising cells supported on a graphene-nanofiber support comprising a nanofiber support coupled with graphene according to the first aspect of the present invention; Tracking said cells by luciferase; And tracking the graphene-nanofiber support by photoacoustic tomography. The present invention also provides a photoacoustic imaging technique for tracking graphene-nanofiber scaffolds.

According to one embodiment of the present invention, the graphene-nanofiber support has a layer-by-layer structure, and has a structure and mechanical strength very similar to those of the brain. Furthermore, since graphene oxide (GO) exhibits hydrophilicity, it has superior dispersibility to carbon-based materials having different dispersibility and is excellent in stability of colloidal property and is easy to handle. In addition, the Young's modulus is about 0.5 Tpa to about 1 Tpa, and it has not only the cell adhesion but also the differentiation promoting effect, and it can be utilized for the cultivation of stem cells, An accelerated effect can be achieved.

In one embodiment herein, bacterial cellulose (BC), which has been used as a scaffold for cell culture, is used as a scaffold for brain cultures because of its high tensions, Transplantation is possible and commercialization is possible. In addition, BC is easy to produce, low in toxicity due to high purity, high in biological suitability, and is used in actual transplantation because transplantation in vivo shows less rejection and immune response. In addition, since BC is a substance produced by bacteria, it is similar to the original extracellular matrix and can be used for cell engraftment without additional chemical modification.

In one embodiment of the present invention, by using GO-BC, which combines GO and BC, the structure of BC is similar to the intracellular microenvironment, thereby enhancing primary cell adhesion and promoting cell differentiation by GO . Since the longitudinal elastic modulus of the GO-BC is very similar to that of the brain, it can be applied to nerve cell engraftment studies. Since the GO and BC do not cause an immune response, More conformance. In addition, depending on the intended use of the GO-BC, it is easy to add a modification and / or a compound to BC or GO, thereby suitably adapting to the targeted cell adhesion. In particular, nanofiber materials in the form of cellulose, which hybridize with graphene, are coated with natural proteins and do not require coating of other extracellular matrix.

The photoacoustic tomography according to one embodiment of the present invention is an image technology that combines the advantages of the existing ultrasonic wave by displaying the image combined with the ultrasound wave, which is capable of imaging up to the deep part of the tissue, as compared with the other image method. The photoacoustic tomography allows cell tracking through luciferase and simultaneous tracking of the grafted support, unlike the prior art, which was only capable of cell tracing through luciferase. In addition, since the graphene-nanofiber scaffold can be traced according to the date by using the photoacoustic tomography, it is highly likely to be used as a contrast agent in the future.

1 is a photoacoustic tomography ultrasound image according to the concentration of different graphene oxide in one embodiment of the present invention.
2 is a graph showing the intensity of photoacoustic signals according to graphene oxide concentration in one embodiment of the present invention.
Figure 3 is a bioluminescent image of a subcutaneously implanted mouse, a cell comprising BC, and a supporter-free cell, comprising GO-BC support in one embodiment of the invention.
Figure 4 is a photoacoustic image of a mouse, a cell comprising BC, and a cell bearing a GO-BC support, and a mouse without a support-less cell, in one embodiment of the present invention.
Figure 5 is a bioluminescent image of a mouse in which the cells comprising the GO-BC support are implanted in the brain injury site, in one embodiment of the present invention.
Figure 6 is a nestin, 200-fold Tuj-1, and 800-fold Tuj-1 antibody immunostaining image, in one embodiment of the invention.
7 (a) to (f) show photoacoustic signals of a cell culture support inserted into a mouse according to one embodiment of the present invention.
8 (a) to 8 (c) are immunostained images of mice transplanted with cells, BC-cells and GO-BC / cell complexes, respectively, in one embodiment of the present invention.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art can easily carry out the present invention. It should be understood, however, that the present invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. In the drawings, the same reference numbers are used throughout the specification to refer to the same or like parts.

Throughout this specification, when a part is referred to as being "connected" to another part, it is not limited to a case where it is "directly connected" but also includes the case where it is "electrically connected" do.

Throughout this specification, when a member is " on " another member, it includes not only when the member is in contact with the other member, but also when there is another member between the two members.

Throughout this specification, when an element is referred to as " including " an element, it is understood that the element may include other elements as well, without departing from the other elements unless specifically stated otherwise. The terms " about ", " substantially ", etc. used to the extent that they are used throughout the specification are intended to be taken to mean the approximation of the manufacturing and material tolerances inherent in the stated sense, Accurate or absolute numbers are used to help prevent unauthorized exploitation by unauthorized intruders of the referenced disclosure. The word " step (or step) " or " step " used to the extent that it is used throughout the specification does not mean " step for.

Throughout this specification, the term " combination (s) thereof " included in the expression of the machine form means a mixture or combination of one or more elements selected from the group consisting of the constituents described in the expression of the form of a marker, Quot; means at least one selected from the group consisting of the above-mentioned elements.

Throughout this specification, the description of "A and / or B" means "A or B, or A and B".

Hereinafter, embodiments and examples of the present invention will be described in detail with reference to the accompanying drawings. However, the present invention is not limited to these embodiments and examples and drawings.

A first aspect of the present invention provides a support for cell culture comprising graphene and nanofibers bonded with the graphene.

In one embodiment of the present invention, the graphene is very stable not only structurally and chemically but also as an excellent conductor. The graphene exhibits excellent conductivity due to its structural characteristics with a relatively small surface defect with a thickness of one atom and can be used as a support for tissue or brain damage since it is not harmful to the living body. For example, the graphene may include, but is not limited to, those formed by chemical vapor deposition (CVD). For example, the graphene may include, but is not limited to, those formed by a Hummer's method or a modified Hummer's method.

In one embodiment of the present invention, the graphene has a large surface area relative to its size, which can mount a high-capacity drug, and can be utilized as an effective drug delivery and photo-thermal treatment material in a cell. And may promote, but not limited to, differentiation of neural progenitor cells and neural stem cells.

In one embodiment of the invention, the graphene may include, but is not limited to, graphene, oxidized graphene, and reduced oxidized graphene.

In one embodiment of the present invention, the cell culture support (graphene-nanofiber support) comprising nanofibers bonded with graphene is a three-dimensional support, which is advantageous for cell infiltration and easy to adsorb cells Dimensional network structure, and may include, but not limited to, those which are handy at the time of transplantation.

In one embodiment of the invention, the nanofibers may include, but are not limited to, cellulose.

In one embodiment of the present invention, the support for cell culture may include, but is not limited to, a combination of graphene and bacterial cellulose (BC).

In one embodiment of the present invention, the BC is used as a scaffold for cell culture. The BC may be used after the addition of the compound and / or protein according to the intended use, but may not be limited thereto. For example, when the BC is phosphorylated, it is possible to cultivate osteoblasts. When the BC is subjected to the sulfation treatment, heat resistance is enhanced. When the BC is subjected to the alkali treatment , And can inhibit the degradation by cellulase. When RGD peptide is treated as a protein in BC, the ability to engage vascular endothelial cells may be strengthened, but the present invention is not limited thereto.

In one embodiment of the present invention, the BC may be similar to a cellular microenvironmental structure of a cell to increase the engraftment of cells and promote differentiation into graphene.

In one embodiment of the present invention, the structure and / or properties of graphene oxide-bacterial cellulose (GO-BC) can be controlled by controlling the amount and / or type of filler contained in the BC. In one embodiment of the present invention, the GO-BC is made of hydrophilic cellulose, but due to its structural form it can also carry hydrophobic materials.

In one embodiment of the present invention, the graphene-nanofiber support may be selected from the group consisting of agarose gel, pH (hydrophilic) 2-hydroxyethyl methacrylate) polymer hydrogel, PLGA but are not limited to, nanofibers including poly (glycolic acid), PGA (poly (glycolic acid)], PCL (polycaprolactone), and the like, .

In one embodiment of the present invention, the graphene-nanofiber support has a layer-by-layer structure, and has a structure and mechanical strength very similar to that of the brain.

In one embodiment of the invention, the graphene-nanofibrous support comprises platelet growth factor (PDGF), epithelial growth factor (EGF), retinoic acid, HA (hydroxyapatite), trichloroacetic acid Or a combination thereof, may be attached, but the present invention is not limited thereto. For example, the attached material may have, but is not limited to, the function of inducing growth of a cell and differentiation into a desired cell as a growth factor and / or a functional factor.

In one embodiment of the present invention, the structure, shape, size, or size of the cell culture support may be adjusted to control the adsorption characteristics of stem cells, but the present invention is not limited thereto. The adsorption characteristics may include, for example, a cell adsorption area, a binding property with a binding receptor present on the cell surface in the cell culture support, or a binding property with an extracellular matrix (ECM) But may not be limited. The difference in the adsorption characteristics of the stem cells may affect the cell signaling system which affects stem cell proliferation and / or differentiation. As a result, it is also possible to regulate the adsorption pattern of the cells, the direction of the cells, the polarity of the cells, the constitution of the intercellular network, and / or the cell differentiation.

According to an embodiment of the present invention, the cell may include, but is not limited to, a stem cell. For example, the stem cell is a cell capable of differentiating into various cells constituting a biological tissue. The stem cell can be obtained from embryonic, fetal, or adult tissues, And may include, but is not limited to, embryonic stem cells, adult stem cells, hematopoietic stem cells, neural stem cells, mesenchymal stem cells, or precursor cells of various somatic cells. For example, the support for stem cell culture comprising the graphene-nanofiber may be a stem cell cultured in vivo, wherein the grafted stem cells are differentiated from the grafted tissue and networked with surrounding cells, But may not be limited to, those which exhibit very good support properties in curing. For example, the support for cell culture containing the graphene-nanofibers is excellent in adhesion to cells, thereby improving cell density and enhancing cohesion between cells, But are not limited thereto. For example, the characteristics of the graphene-nanofiber contributes to forming a network with grafted tissues so that the grafted stem cells can perform their functions sufficiently at the graft site, and to suppress the washing phenomenon at the graft site But are not limited thereto.

According to one embodiment of the present invention, the stem cells may include embryonic stem cells or adult stem cells, but the present invention is not limited thereto. For example, the embryonic stem cells may be cells obtained by culturing the inner cell mass of a developing blastocyst, and may include self-replicating cells and cells capable of differentiating into cells in vivo , But may not be limited thereto. For example, the adult stem cells may be self-replicating as embryonic, fetal, or adult stem cells, but have a limited ability to differentiate from embryonic stem cells so that they can differentiate into specific types of cells, But are not limited to, precursor cells.

According to an embodiment of the present invention, the adult stem cells may be hematopoietic stem cells, neural stem cells, or mesenchymal stem cells, . For example, the hematopoietic stem cells may have the ability to produce fully differentiated blood cells, such as red blood cells, white blood cells, mononuclear cells, megakaryocytes, natural killer cells, T lymphocytes, or B lymphocytes, Cells, but may not be limited thereto. For example, the neural stem cells are capable of producing fully differentiated neurons, for example, neurons, astrocyte, or glial cells, But the present invention is not limited thereto. For example, the mesenchymal stem cells have self-replicating ability and have the ability to produce completely differentiated muscle cells, adipocytes, nerve cells, bone cells, etc. Specifically, the mesenchymal, white adipocytes, But are not limited to, cells that have the ability to produce cells, fibroblasts, neurons, astrocytes, glial cells, osteoblasts, osteoclasts, or cartilage cells, .

A second aspect of the present application provides a method for producing stem cells, comprising culturing stem cells on a cell culture support comprising a graphene-nanofiber support comprising a nanofiber support coupled with graphene according to the first aspect of the present invention, Thereby providing a method of culturing the cells. The second aspect of the present invention relates to a method for culturing stem cells on a cell culture support according to the first aspect of the present invention, and a detailed description thereof is omitted for the parts overlapping with the first aspect of the present application. However, The description of the aspects may be applied equally to the second aspect of the present invention even if the description thereof is omitted.

In one embodiment of the present invention, the stem cell is a cell capable of differentiating into various cells constituting a biological tissue, and is a cell capable of differentiating into a cell obtainable from each tissue of an embryo, fetus, or adult body Stage undifferentiated cells, but the present invention is not limited thereto.

In one embodiment of the present invention, culturing the stem cells efficiently differentiates the stem cells into specific cells, specifically, neurons, bone cells, chondrocytes, muscle cells, brain cells, or adipocytes But is not limited thereto. For example, the method for culturing the stem cells may include, but is not limited to, preparing a cell and an extracellular support for in vivo transplantation for the purpose of treating diseases.

In one embodiment of the present invention, culturing the stem cells may include, but is not limited to, regulating the growth or differentiation of the stem cells. For example, the growth of the stem cells can be accomplished by culturing the stem cells in an adhesion culture and / or suspension culture (hereinafter, referred to as " growth culture ") in a growth medium supplemented with a suitable growth factor and / ) To induce the proliferation of stem cells by inhibiting the differentiation of stem cells and inducing self-replication, but the present invention is not limited thereto. For example, the regulation of the differentiation of stem cells can be accomplished by differentiating the stem cells from self-replication of stem cells by coalescence and / or suspension culture of the stem cells in a differentiation medium supplemented with appropriate growth factors and / or serum on a liquid medium Including, but not limited to, induction of cell growth.

In one embodiment of the present invention, the stem cells may be embryonic stem cells or adult stem cells, and the adult stem cells may be hematopoietic stem cells, neural stem cells, mesenchymal stem cells, or precursors of various somatic cells But is not limited thereto. For example, the embryonic stem cell may be a cell cultured by isolating an inner cell mass of a developing blastocyst, and may include cells having self-replication ability and ability to differentiate into all cells in vivo , But may not be limited thereto. For example, the adult stem cells are self-replicating stem cells present in an embryo, a fetus, or an adult, but have a limited ability to differentiate from embryonic stem cells, so that cells that can only differentiate into specific types of cells are included , But may not be limited thereto.

In one embodiment of the present invention, the adult stem cells may include hematopoietic stem cells, neural stem cells, or mesenchymal stem cells, but the present invention is not limited thereto. For example, the hematopoietic stem cells are capable of producing fully differentiated blood cells such as red blood cells, white blood cells, mononuclear cells, megakaryocytes, natural killer cells, T lymphocytes, or B lymphocytes, But are not limited thereto. For example, the neural stem cells are capable of producing fully differentiated neurons, for example, neurons, astrocyte, or glial cells, But the present invention is not limited thereto. For example, the mesenchymal stem cells have self-replicating ability and have the ability to produce completely differentiated muscle cells, adipocytes, nerve cells, bone cells, etc. Specifically, the mesenchymal, white adipocytes, But are not limited to, cells that have the ability to produce cells, fibroblasts, neurons, astrocytes, glial cells, osteoblasts, osteoclasts, or cartilage cells, .

According to one embodiment of the present invention, the method of culturing the stem cells may further include a step of flowing an electric current to the graphene-nanofibrous supporter, but the present invention is not limited thereto. For example, in the step of flowing current to cells induced to differentiate into neurons, the calcium ion channel in the cell membrane of the neuron is opened, and calcium ions accumulate inside the neuron. Therefore, by accumulating calcium ions, Including, but not limited to, the ability to detect successful differentiation of cells.

A third aspect of the present invention provides a stem cell cultured on a graphene-nanofiber support by the method of cultivation of the second aspect of the present application. A third aspect of the present invention relates to a stem cell cultured on a support for cell culture according to the first aspect of the present application by a culturing method according to the second aspect of the present application, wherein the stem cell which overlaps with the first aspect or the second aspect of the present invention The description of the first aspect or the second aspect of the present invention can be applied equally to the third aspect of the present invention.

In one embodiment of the present invention, the stem cell is a cell capable of differentiating into various cells constituting a biological tissue, and is a cell which can be obtained from embryonic, fetal, or adult tissues, And may include, but not limited to, embryonic stem cells, adult stem cells, hematopoietic stem cells, neural stem cells, mesenchymal stem cells, or various somatic precursor cells. Specifically, the stem cells may include, but are not limited to, somatic precursor cells or adult stem cells in the process of being cultured on a graphene-nanofiber support to differentiate into somatic cells. More specifically, the stem cell may be a neural stem cell, a precursor of a neuron cell, a hematopoietic stem cell, a precursor of a blood cell, a mesenchymal stem cell, a precursor of a bone cell, a precursor of a cartilage cell, a precursor of an adipocyte, But may not be limited thereto.

A fourth aspect of the invention is a cell therapy composition for treating brain diseases comprising cells supported on a graphene-nanofiber support comprising a nanofiber support coupled with graphene according to the first aspect of the present application . The fourth aspect of the present invention relates to a composition for cell therapy for the treatment of brain diseases, comprising cells supported on a support for cell culture according to the first aspect of the present invention, wherein parts overlapping with the first aspect of the present invention The description of the first aspect of the present invention can be similarly applied even if the description thereof is omitted in the fourth aspect of the present application.

In one embodiment of the present invention, the composition for cell therapy comprises a mixture of the graphene-nanofiber support and various biopolymers, or a mixture of the graphene-nanofiber support and growth factors and / or differentiation promoting factors But it may not be limited thereto.

In one embodiment of the present invention, the composition for cell therapy may be inserted directly into a site to be treated using a syringe, inserted through surgery, or inserted through a circulatory system, but is not limited thereto. For example, the graphene-nanofiber scaffold may comprise a network of grafted tissues so that the grafted cells can perform their function well at the graft site, and inhibiting the wash away at the graft site But may not be limited thereto. For example, the composition for cell therapy, which comprises cells supported on the graphene-nanofiber support, has an advantage of being easily injected at a desired position by injection to minimize other side effects caused by surgery, But may not be limited thereto. For example, the injected cell therapy composition itself forms a structure over time, and the structure is suitable for forming an intercellular network, and is suitably applied through electrical induction due to the electrical conductivity of the graphene Including, but not limited to, the ability to move to a desired location and to act as a support with the cells fluidly at the site where the tissue is broken. For example, the cells contained in the composition for cell therapy can be used to replace damaged cells of the grafted tissue in vivo to heal tissue damage, or to connect grafted tissue with the graft-nanofiber scaffold Reconstituting the tissue, and so on. For example, the composition for cell therapy according to the fourth aspect of the present invention may contain a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by those skilled in the art. , Or may be manufactured by inserting it into a multi-dose container, but the present invention is not limited thereto. The formulations herein may be in the form of solutions, suspensions, oil-in-water or water-in-oil liquid emulsions, elixirs, syrups or emulsions in aqueous or non-aqueous liquids and may additionally comprise dispersants or stabilizers, have. For example, pharmaceutical compositions for parenteral administration include, but are not limited to, dispersions, suspensions, emulsions, or sterile injectable solutions prior to use.

In one embodiment of the present invention, the cells may include, but are not limited to, cells cultured by a method of culturing stem cells comprising culturing stem cells on the graphene-nanofiber support .

According to an embodiment of the present invention, the cell may be a hematopoietic stem cell, a neural stem cell, a mesenchymal stem cell, a precursor cell of various somatic cells, a nerve cell, a bone cell, a cartilage cell, a muscle cell, However, the present invention is not limited thereto. For example, the hematopoietic stem cells are capable of producing fully differentiated blood cells such as red blood cells, white blood cells, mononuclear cells, megakaryocytes, natural killer cells, T lymphocytes, or B lymphocytes, But are not limited thereto. For example, the neural stem cells are capable of producing fully differentiated neurons, for example, neurons, astrocyte, or glial cells, But the present invention is not limited thereto. For example, the mesenchymal stem cells have self-replicating ability and have the ability to produce completely differentiated muscle cells, adipocytes, nerve cells, bone cells, etc. Specifically, the mesenchymal, white adipocytes, But are not limited to, cells that have the ability to produce cells, fibroblasts, neurons, astrocytes, glial cells, osteoblasts, osteoclasts, or cartilage cells, .

A fifth aspect of the invention herein is a method of treating a cell, comprising administering to the animal a composition for cell therapy comprising cells supported on a graphene-nanofiber support comprising a nanofiber support coupled with graphene according to the first aspect of the invention step; Tracking said cells by luciferase; And tracking the graphene-nanofiber support by photoacoustic tomography. The present invention also provides a photoacoustic imaging technique for tracking graphene-nanofiber scaffolds. A fifth aspect of the present invention is a method for treating a brain disease comprising administering to a animal a composition for cell therapy for treating a brain disorder comprising cells supported on a cell culture support according to the first aspect of the present invention, The detailed description of the parts overlapping with the first aspect of the present application is omitted. However, the description of the first aspect of the present invention can be similarly applied even if the description is omitted in the fifth aspect of the present application.

In one embodiment of the present invention, the composition for cell therapy comprises a mixture of the graphene-nanofiber support and various biopolymers, or a combination of the graphene-nanofiber support and growth factors and / or differentiation promoting factors Mixed, but may not be limited thereto.

In one embodiment of the present invention, the composition for cell therapy may be administered to an animal by inserting a syringe or the like directly through a surgical site, or through a circulatory system, .

In one embodiment of the present invention, the support for cell culture comprising the graphene-nanofibers prepared according to the first aspect of the present application is applied to an actual brain injury model, and the cell culture support comprising the graphene- Survival of the transplanted cells in the supporter can be monitored in vivo using non-invasive optical imaging techniques to confirm the technique of treating the disease of the supporter. In addition, it is possible to track the support by time using a photoacoustic tomography method in order to track the support in a three-dimensional form transplanted in a living body, and it can also be used as a contrast agent, thereby determining the resolution of the support .

In one embodiment of the present invention, among the cell therapy compositions administered to the animal, the cells are traceable by luciferase.

In one embodiment of the present invention, due to the light absorption properties of the graphene, the graphene exhibits a strong signal during photoacoustic tomography, so that the graphene-nanofibre scaffold in which the cells are cultured can also be traced.

Hereinafter, embodiments of the present invention will be described in detail. However, the present invention is not limited thereto.

[ Example  One]

Phantom production of PAT in vivo

A phantom for photoacoustic imaging was prepared as follows: 1.5% agarose gel in a tris borate ethylenediaminetetraacetic acid (TBE) buffer was poured into a 20 cm x 10 cm square frame, lt; RTI ID = 0.0 > holes. < / RTI > After clotting, 96-well PCR plates were removed and the resulting holes filled with different concentrations of 20 μL of graphene oxide ranging from 1.25 mg / mL to 10 mg / mL diluted with 1.5% agarose gel.

GO-BC cell manufacturing

To prepare a cell culture support (graphene-nanofiber support) containing graphene-nanofibers, graphene oxide (GO) and bacterial cellulose (BC) were hybridized. Specifically, a graphene-nanofiber support was prepared by dispersing graphene oxide in a culture medium in which bacterial cellulose was biosynthesized. At this time, the graphene oxide grains were prepared by Hummer's method, and the graphene-nanofiber support was washed more than 100 times before the graphene-nanofiber support was manufactured.

The GO-BC preserved in isopropyl alcohol was imprinted with a 1 cm-diameter puncher for use in an appropriate size and then washed with a sufficient amount of PBS to remove isopropyl alcohol. 24-well and then load the GO-BC to the plate, and included a 10 μL of resuspended with ⁵ x 10 5 F3-effLuc cells in PBS on a GO-BC. The cells / GO-BC complexes were then incubated for 30 minutes at 37 ° C for internalization of the cells, followed by the addition of 1 mL of cell culture medium.

In vivo luminescence enzyme analysis luciferase  assay and in vivo bioluminescence Imaging

In vivo luminescence enzyme assays were performed using luminescence enzyme kits (Applied Biosystems, Carlsbad, Calif., USA). F3-effLuc cells were seeded in 24-well or 6-well plates, and 24 hours after plating, the cells were washed with PBS and lysed using lysis buffer. The dissolved F3-effLuc cells were transferred to 96-well plates for the detection of bioluminescence signals. The luminescence enzyme strength was measured using a Varioskan Flash (Thermo Fisher Scientific, Vantaa, Finland) at a time of one second. For in vivo imaging, GO-BC was washed with PBS. For the transplantation of cells / GO BC-complex, ⁵ x 10 5 F3-effLuc cells were obtained using trypsin and resuspended in PBS. After 10 μL of resuspended F3-effLuc cells were included in the GO-BC support and the F3-effLuc / GO-BC complex was incubated for 30 minutes, it was injected into the cavity of the cerebral cortex ) (N = 3). To obtain bioluminescent images, the mice fed a sedative containing 2% isoflurane in 100% O ₂ via nasal cones. _D -luciferin (Caliper Life Sciences, Hopkinton, MA, USA) was diluted to 3 mg / 100 μL in saline, and _D -luciferin 0.6 mg was injected into the brain at 0, 1, 2, 4, 8, ≪ / RTI > Cyclosporin A (5 mg / kg) was administered intraperitoneally daily after transplantation to inhibit the innate immune response against human F3-effLuc cells. The IVIS-100 imaging system (Caliper Life Science) equipped with a CCD camera was used for in vivo bioluminescence imaging. The image was obtained by integrating light for 5 minutes. The emission intensity of the region of interest from each image was quantified to test the survival rate of the transplanted cells.

In vivo of GO-BC transplanted mice Photoacoustic Imaging

Seven adult male balb / c-nu mice weighing 30 g to 40 g were purchased from OrientBio (Sungnam, Kyungki, Korea). After 1.5-2 cm incision of the skin, the GO-BC / cell complex was injected subcutaneously under the thigh of the mouse and finally the incised skin was sutured. In addition, BC / cell complex and F3-effLuc cells without support were transplanted into control. Photoacoustic imaging was performed with a Vevo LAZR System (VisualSonics Inc., Toronto) with a wavelength of 700 nm and a photoacoustic intensity of 40 dB. The area of photoacoustic imaging was abundant with ultrasound gels, and Aqua Sonic (Parker, NJ, USA) and photoacoustic / ultrasound probes were contacted to the imaging site for visualization of tomographic images.

Cerebral Corticalectomy and Animal Model

A 5-week-old adult male Sprague-Dawley (SD) albumin mouse weighing 180 g to 200 g was provided by a clinical trial laboratory at Seoul National University Hospital. Mice were anesthetized with Zoletile 50 (75 mg / kg, ip) and xylazine (10 mg / kg, ip). Anesthetized mice were placed in a stereotactic frame and height was controlled with lambda and bregma. The midline scalp and temporalis were incised, and the exposed skull was removed with a hand drill using a hand drill at the following coordinates: A) 4 mm posterior and 1 mm lateral, B) 2 mm posterior and 1 mm lateral, C ) 4 mm lateral and 6 mm lateral. After removal of the skull, the exposed cranial and motor cortex were removed by the surgical blade, and the ablated area was filled with gel foam (Pharmacia and Upjohn, Kalamazoo, MI). Finally, the incised skin was sutured.

Brain amputation and histological analysis

Anesthetized mice were sprayed with gentle perfusion with saline containing heparin and 4% paraformaldehyde (PFA; Sigma-Aldrich). The mouse brain was removed, first fixed in 4% paraformaldehyde for 24 hours, and then dried in 10%, 20%, and 30% sucrose at 4 占 폚. The specimens were frozen in OCT medium (Leica, IL, USA) and successive sections of 14 μm thick coronal surfaces were cut and attached on gelatin-coated slides. One brain section in each group underwent basic hematoxylin and eosin (H & E) staining, and six sets of sections were immunohistochemically stained. Portions on the glass slide were osmoticized in PBS containing 0.5% Triton-X for 5 minutes at 4 ° C, rinsed 3 times with PBS for 5 minutes, then incubated in 1% normal horse serum for 1 hour at room temperature. Slides were incubated in a 1: 500-diluted aqueous solution of anti-LucJ (Millipore Co., Billerica, MA, USA), anti-luciferase (Millipore Co.) for one day. The location of transplanted NSCs was observed by staining with anti-Thy1.1 and anti-luciferase antibodies. Immunohistochemistry was performed using a confocal laser microscope (LSM 510; Carl Zeiss, Jena, Germany).

[ Example  2]

Cerebral cortex resection  Mouse model and F3- effluc / GO-BC implant

Seven adult male Sprague-Dawley (SD) mice weighing 200 g to 250 g were purchased from OrientBio (Sungnam, Kyungki, Korea). Rats anesthetized by mixing zoletil 50 (15 mg / kg, ip) and Rompun (10 mg / kg, ip) were placed in a stereotactic frame to accurately indicate the surgical coordinates. After the incision of the midline scalp and periosteum, the skull was positively removed with an electric hand drill at the following coordinates: a) 4 mm side / 1 mm side, b) 2 mm tail side / 1 mm side, and c) 4 mm side / 6 mm side. After the triangular skull was removed, the exposed dura mater and motor cortices were carefully excised with a surgical knife, and the bipolar electrode was used for hemostasis. Finally, the cavity was filled with an absorbable hemostatic gelatin sponge (Ethicon, Somerville, NJ, USA). The incised skin was completely hemostatic and then sutured. All animal experiments were approved by the Animal Care and Use Committee of Seoul National University Hospital (IACUC NO.13-2013-001-8). For cell / support transplantation, sterile BC or GO-BC support on ethanol was extensively washed with PBS for complete removal of ethanol. For hapip (incorporation) of the cell / support complex, ⁵ x 10 5 F3-effluc cells and recovered using put stream, it was re-suspended in PBS. The resuspended F3-effluc / support complex of 10 [mu] m was then seeded into the GO-BC support. After incubation of the F3-effluc / support complex for 2 hours, the F3-effluc / support complex was grafted into the hole of the cerebral cortical resected mouse brain.

Photoacoustic  Obtaining images

Phantoms for photoacoustic imaging were prepared as follows; A 1.5% agarose gel in TBE buffer was poured into a 20 cm x 10 cm square mold and a 96-well PCR plate was inserted to create holes. After clotting, 96-well PCR plates were removed and the resulting holes filled with different concentrations of graphene oxide in a volume of 20 μL ranging from 1.25 mg / mL to 10 mg / mL diluted with 1.5% agarose gel. As a GO-BC / cell complex-injected mouse for in vivo photoacoustic imaging, adult male BLAB / c-nu mice weighing 30-40 mg were purchased from OrientBio (Sungnam, Kyungki, Korea). After 1.5 cm to 2 cm incision of the skin, the GO-BC / cell complex was injected subcutaneously into the thigh of the mouse and finally the incised skin was sutured. The supernatant-free BC / cell complex and F3-effLuc cells were also implanted for control. Photoacoustic imaging was performed with a Vevo LAZR system (VisualSonics Inc., Toronto, Canada) at an excitation wavelength of 700 nm and a photoacoustic intensity of 40 db. The area for photoacoustic imaging was abundant with ultrasound gels, and Aqua Sonic (Parker, NJ, USA) and photoacoustic / ultrasound probes were contacted to the imaging site for visualization of tomographic images.

result

For the phantom study of Example 1 above, different concentrations of graphene oxide ranging from 1.25 mg / mL to 10 mg / mL were located ~ 5 mm below the surface of the agarose gel phantom, as shown in FIG. In the ultrasonic image, the white region indicates the position and shape of graphene oxide in the gel phantom, and the photoacoustic signal is easily detected in concentration proportion from graphene oxide. The ROI value was measured and the photoacoustic signal was proportional to the increased concentration of graphene oxide (FIG. 2).

Cells without GO-BC / cells, BC / cells, and supporters were implanted subcutaneously in hairless mice. The distribution of transplanted cells was visualized as bioluminescence images on days 0, 2, 4, 8, 10, and 12 (FIG. 3). As shown in Fig. 4, the ultrasonic image showed the position and shape of each support. In the photoacoustic mode, the group to which the GO-BC / cell complex was implanted exhibited a high photoacoustic signal along the edge of the GO-BC. In contrast, photoacoustic signals did not appear in both transplanted cells without support and in groups transplanted with BC / cell complex cells.

1 day, 2 days, 4 days, 8 days, 10 days, and 12 days after transplantation of GO-BC / cell, BC / cell, and supporter- (Fig. 5).

In order to confirm the differentiation of neural stem cells in the GO-BC / cell complex, immunohistochemical staining of the brain tissue grafted with GO-BC / cell complex through Nestin and Tuj-1 antibodies, which are used as neural differentiation markers, (Fig. 6).

It is known that the properties of graphene oxide with strong NIR absorption capability make it possible to obtain photoacoustic signals. This phenomenon suggested the possibility of in vivo tracing of implanted GO-BC scaffolds. As in Example 2 above, the photoacoustic signal in the damaged brain region of the GO-BC / cell graft was accurately observed even if a weak background signal was present around the implanted portion (Fig. 7 (d)). On the other hand, photoacoustic signals were not found in the group treated with BC / cells and cells alone (Fig. 7 (c) and (f)).

Human-derived neural stem cells (F3), BC scaffolds / neural stem cells, GO-BC scaffolds / neural stem cells were transplanted into brain-damaged mice from which the motor cortex was removed, The tissues were excised and immunostained using brain cell specific MAP2 antibody. As shown in Figs. 8 (a) to 8 (c), it was confirmed that the neural stem cells survived in the cell-only transplanted group and the expression of MAP2, which is a neural differentiation marker, was very small )]. In the BC support / neural stem cell group shown in FIG. 8 (b), the cell survival rate was slightly higher than that of the cell-injected group, and it was confirmed that it was differentiated into some MAP2 positive neurons. As shown in FIG. 8 (c), in the group transplanted with the GO-BC / neural stem cell complex according to Example 2, the survival rate of the transplanted cells was very high and most of the cells were differentiated into neurons I could confirm.

We have invented an implantable hybrid graphene-bacterial cellulosic (GO-BC) support to demonstrate a GO-BC support as a potential regenerative nanomaterial by applying it to a mouse model of brain injury. In addition, the support-effect of the GO-BC support was evidently demonstrated from corticectomized mice by continuously observing the survival of transplanted neural stem cells contained in the GO-BC support, The development of the GO-BC support was directly confirmed by photoacoustic imaging.

It will be understood by those of ordinary skill in the art that the foregoing description of the embodiments is for illustrative purposes and that those skilled in the art can easily modify the invention without departing from the spirit or essential characteristics thereof. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. For example, each component described as a single entity may be distributed and implemented, and components described as being distributed may also be implemented in a combined form.

The scope of the present invention is defined by the appended claims rather than the detailed description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included within the scope of the present invention.

Claims (11)

Graphene; And
The graphene-
≪ / RTI >
The method according to claim 1,
Wherein the nanofiber comprises cellulose.
The method according to claim 1,
Wherein the cell comprises stem cells.
The method of claim 3,
Wherein the stem cell comprises embryonic stem cells or adult stem cells.
5. The method of claim 4,
Wherein the adult stem cells comprise hematopoietic stem cells, neural stem cells, or mesenchymal stem cells.
A method of culturing stem cells, comprising culturing stem cells on a graphene-nanofiber support comprising a nanofiber support coupled with graphene according to any one of claims 1 to 5.
The method according to claim 6,
Wherein culturing the stem cells comprises regulating growth or differentiation of the stem cells.
A stem cell cultured on a graphene-nanofiber support according to the method of claim 6.
A cell therapy composition for treating brain diseases, comprising cells supported on a graphene-nanofiber support comprising a nanofiber support in combination with the graphene according to any one of claims 1 to 5.
10. The method of claim 9,
Wherein said cells comprise cells cultured by a method for culturing stem cells comprising culturing stem cells on a graphene-nanofiber support.
6. A method for treating a cell, comprising administering to a animal a composition for cell therapy comprising cells supported on a graphene-nanofiber support comprising a nanofiber support coupled to graphene according to any one of claims 1 to 5;
Tracking said cells by luciferase; And
Tracking the graphene-nanofiber support by photoacoustic tomography
/ RTI > photoacoustic imaging technique for tracking graphene-nanofiber scaffolds.

Images