KR101539822B1 - Method for preparing concentrated cell culture medium via 3D-culture and use thereof - Google Patents

Abstract

The present invention relates to a method for producing a culture medium containing a growth factor or cytokine, which comprises culturing adult stem cells on a scaffold and culturing the same in a three-dimensional manner, a concentrated culture liquid prepared by the above method, A preparation for bone regeneration regenerating composition comprising a culture fluid-loaded scaffold, which comprises the step of preparing the culture solution, and the step of loading the prepared culture solution into a scaffold for grafting. And a transplantation scaffold in which a culture solution prepared by the above method is loaded.

Description

Method for preparing high-concentration culture medium by 3-dimensional culture of stem cells and use of high-concentration culture medium {Method for preparing concentrated cell culture medium via 3D-culture and use thereof]

The present invention relates to a method for producing a culture medium containing a growth factor or cytokine, which comprises culturing adult stem cells on a scaffold and culturing the same in a three-dimensional manner, a concentrated culture liquid prepared by the above method, A method for producing a bone graft regenerating implant comprising the step of loading the composition into a grafting scaffold and a bone graft regeneration implant comprising the grafting scaffold prepared by the method .

Bone tissue can be damaged by various diseases such as spinal injuries, fractures, infections, and tumors. In the case of simple fracture, natural treatment is possible, but a therapeutic agent for treating and / or regenerating bone loss in excess of the natural treatment limit is needed. In addition, when extracted due to periodontal disease or trauma, alveolar bone is absorbed, which causes problems in prosthetic treatment and implant treatment. In particular, for successful implants, the implant of a certain length and diameter should be placed. However, if the alveolar bone is insufficient, implant placement may be limited. In these areas, bone grafting is required to increase the height and width of the alveolar bone, and several regenerative treatments have been developed for this purpose. The most widely used bone defects are bone grafts, and autogenous bone, allogeneic bone, heterogeneous bone, or synthetic bone is used as a graft material for bone regeneration. Although autogenous bone contains bone cells capable of direct bone formation and is the best bone graft material with no rejection, it can guarantee a high success rate, but it requires additional operation on the harvesting site for bone harvesting, It is not easy to collect as much as necessary because the amount is limited. Therefore, in clinical practice, there is an increasing demand for the development of bone graft substitutes for replacing autogenous bone. Allografts are available as a substitute for autogenous bone because they can be harvested in large quantities compared to autogenous bone grafts, have the advantage of being transplanted with one operation, and have a high possibility of osteogenesis. However, it is not easy to manipulate, does not show consistent bone regeneration results, may cause failure in bone regeneration, side effects such as immune rejection may occur, and bone fragments obtained from disease holders may cause disease transmission This is controversial. Bone marrow is widely used to treat bone tissue from non-human animals and to use it as a graft material. The deproteinization process is used to remove the antigen and the possibility of infection of the heterogeneous bone, and the bones that have undergone this deproteinizing process are widely used for the bone grafting in the dental field. In addition, various synthetic materials that can be used as synthetic cores have been developed. To be used as a synthetic bone, it has no toxicity, is biocompatible, is porous, is able to penetrate regenerated bone cells, stably attaches and proliferates, and can be decomposed in the body over time.

On the other hand, with the development of stem cell isolation and culture techniques, a method of transplanting stem cells capable of differentiating into various tissues into a lesion has been used as a cell therapy agent. The cell therapy agent should also satisfy conditions similar to those of the bone graft material. After transplantation, the cells should exhibit the specific functions of the desired cells in the body, be safe, have no immune rejection, and have large enough cells available for transplantation therapy. Therefore, it should be used as an autologous transplantation or as a tissue-compatible form in case of allograft. As such, the cell therapy agent can be classified into autologous, allogeneic and xenogeneic cell therapy depending on the origin of the cell. The autologous cell therapy agent can be used to separate the cells from the patient, to simply proliferate in an undifferentiated state in the test tube, or to induce differentiation and development while culturing, or to change the trait of the cell by the necessary manipulation to impart a specific function, Transplant. On the other hand, the allogeneic cell therapy agent is a cell therapy agent that undergoes a process similar to that of autologous cells, isolates the cells from other healthy donors, transplants in vitro, and transplants them into patients. Cells.

Of these, the most easily performed is an allogenic graft using a homologous cell therapeutic agent. However, the greatest disadvantage of allografts is the immune rejection caused by the transplantation of cells from other individuals. In cases where autotransplantation is possible (bone marrow, epithelium, hematopoietic stem cell), if this is impossible or difficult, it will inevitably carry out allogeneic transplantation, but the application of stem cells is the most limiting factor, and stem cells This is an issue that must be solved to expand the application. Nonetheless, homologous transplantation of adult stem cells has been used as an alternative to overcome the problems of autologous transplantation. It is possible to alleviate the burden on the patient such as the time limit for the in vitro culture after the harvesting, which is a necessary process for autotransplantation, and the economic constraints due to the high cost of the transplant and the operation procedure to be preceded. Research and development are under way to eliminate the risk of possible immune rejection, though it is insignificant. At present, it is based on the injection into differentiated state by mainly differentiating through in vitro culture on the assumption of transplantation in a tissue-compatible form . However, the transplantation into these differentiated cell types blocks the possibility of differentiation into various cells, which is a fundamental advantage of stem cells, and the secretion of cytokines capable of inducing cell activation in the peripheral host system is significantly lowered. And the ability to activate or degrade the electrochemical device is degraded.

Therefore, the present inventors have made intensive researches to find a method of regenerating bone tissue efficiently without worrying about the immunodeficiency reaction by combining the merits of bone graft material and stem cell therapeutic agent transplantation. As a result, stem cells were attached to a 3D porous scaffold, And a biocompatible scaffold loaded with a concentrate of a culture medium containing a large amount of a cytokine obtained by the transplantation of the stem cells.

One object of the present invention is to provide a method for producing a culture medium containing growth factors or cytokines, comprising the step of adhering adult stem cells to a scaffold and culturing in a three-dimensional manner.

It is another object of the present invention to provide a concentrated culture liquid prepared by a method for producing a culture medium containing growth factors or cytokines, which comprises culturing adult stem cells on a scaffold and culturing the same three-dimensionally.

It is still another object of the present invention to provide a composition for regenerating bone tissue comprising the concentrate as an active ingredient.

It is still another object of the present invention to provide a method for producing a culture medium containing a growth factor or a cytokine, which comprises culturing adipose stem cells on a scaffold and culturing the same three-dimensionally, And a step of loading the prepared culture liquid into a scaffold for grafting, wherein the scaffold for grafting is loaded with a culture medium.

It is still another object of the present invention to provide an implant for bone regeneration comprising a scaffold for implantation in which the prepared culture fluid is loaded.

In order to achieve the above object, the present invention provides a method for producing a culture medium containing growth factors or cytokines, comprising culturing adipose stem cells on a scaffold and culturing the same three-dimensionally.

As used herein, the term "adult stem cell" refers to a cell having the ability to differentiate into various cells through self-propagation, which is characteristic of general stem cells, It is a cell with potential ability to develop into a cell with specialized functions and can be separated from bone marrow, skeletal muscle, adipose tissue, umbilical cord blood, umbilical cord, skin, amnion, placenta and the like. In particular, mesenchymal stem cells are cells that aid in the formation of tissues such as cartilage, bone, fat, bone marrow stroma, muscle, and nerves. Although they remain in bone marrow in adults, they also exist in cord blood, peripheral blood, And means cells obtainable therefrom. Preferably, the adult stem cells may be bone marrow derived mesenchymal stem cells, but are not limited thereto. The method for obtaining the adult stem cells can be performed by a method known in the art and is not limited to the method of the embodiment of the present invention.

The term "scaffold " of the present invention means a solid support having a constant skeleton, and includes, without limitation, any stem cell capable of attaching to the scaffold and being able to be proliferated and cultured. Preferably, the scaffold may be a collagen scaffold, but is not limited thereto. Such a collagen scaffold can be used as a porous solid support.

The term "three-dimensional (3D) culture" of the present invention refers to a method in which a three-dimensional culture is adhered to a three-dimensional porous solid support Means that the cells are cultured in three dimensions while filling the pores in the support as the cells proliferate. In a specific embodiment of the present invention, a collagen scaffold was used as a solid support, but it is not limited thereto. Since the method of the present invention uses a small amount of culture medium that can not be obtained in the two-dimensional culture of existing stem cells through such three-dimensional culture, a concentrated growth factor or cytokine can be obtained at a low concentration time have.

The term "growth factor" of the present invention means a naturally occurring substance capable of promoting cell growth, proliferation and differentiation. It is generally a protein or steroid hormone and plays an important role in the regulation of various cellular processes. They serve as signaling molecules between cells, for example, cytokines and hormones that bind to specific receptors on the surface of target cells. Sometimes they promote differentiation and development depending on the type of growth factor. For example, bone morphogenic protein promotes osteoblast differentiation while fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) are involved in vascular differentiation angiogenesis. Though often mixed with cytokines, cytokines are involved in hematopoietic and immune cells. Growth factors have a positive effect on cell division, whereas cytokines are neutral terms on the effect of molecules on proliferation. Preferably, the growth factor is selected from the group consisting of insulin-like growth factor-1 (IGF-1), transforming growth factor-beta1, basic fibroblast growth factor (bFGF), bone morphogenetic protein 2 (BMP2) endothelial growth factor).

The term "cytokine" of the present invention is a small cell-signaling protein molecule secreted in many cells, and collectively refers to signaling molecules widely used for intercellular signal communication. Proteins, peptides or glycoproteins, and the term cytokine includes all of the large and diverse family of regulatory factors produced by the whole human body by cells of various embryological origins. However, it is generally used to refer to an immunomodulating agent such as interleukin and interferon. Preferably, the cytokine may be, but is not limited to, CXCL2 (chemokine CXC motif ligand 2) or IL-8 (interleukin-8).

The term "culture medium" of the present invention means a solution in which stem cells are cultured for a predetermined period of time in a liquid medium which enables growth and survival of the isolated stem cells to be maintained in vitro, And proteins such as cytokines and nutrients remaining in the cell culture. The culture medium includes all conventional culture media suitable for culturing adult stem cells, and the culture medium and culture conditions can be selected depending on the type of cells. The medium used for the culture is preferably a cell culture minimum medium (CCMM), and generally includes a carbon source, a nitrogen source and a trace element component. The minimum culture medium for cell culture is, for example, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI1640, F-10, F- But are not limited to, GMM (Glasgows Minimal Essential Medium) and IMDM (Iscove's Modified Dulbecco's Medium). In addition, the medium may be supplemented with antibiotics such as penicillin, streptomycin or gentamicin, or serum additives such as fetal bovine serum (FBS) and human serum albumin As shown in FIG.

The present invention may further include a step of collecting and concentrating the culture liquid. The step of collecting the culture liquid may be carried out by a method known in the art, for example, centrifugation. Most of the collected cultures are liquid components, which are bulky and thus are not easy to store. Thus, additional concentration steps can be performed. The concentrated culture liquid can be kept in a frozen state by being dispensed, and it can be thawed and diluted to a desired concentration when necessary. Concentration of the protein can be performed by methods known in the art, for example, using a protein concentration filter. In a specific example of the present invention, a protein concentration filter was used to maintain a protein content, while a volume of 1.5 ml of the culture medium was reduced to 30 μl, thereby obtaining a concentrated culture medium in which the concentrations of growth factors and cytokines were significantly increased.

The present invention may further comprise the step of subjecting to and cultivating an electrical stimulus. The above process may be performed by a method known in the art per electric stimulation to promote the secretion of growth factors and cytokines. Preferably, an electrode is provided on a cell culture dish to allow cells to adhere on the electrode, But it is not limited thereto.

The present invention may further include the step of collecting and concentrating the culture medium of the cells cultured in the same manner as described above by applying the electric stimulus.

In the specific examples of the present invention, it was confirmed that gene expression and secretion of osteoclast-related growth factor and stem cell migration-promoting cytokine remarkably increased only by attaching to a collagen scaffold and culturing in three dimensions 2 to 4, Fig. 4 and Fig. 5). Furthermore, by concentrating using a protein concentration filter, it is possible to concentrate the protein to a concentration about 50 times higher than that of the protein (Table 2) (Example 5). The concentrated culture thus prepared exhibits excellent induction of differentiation into bone cells (Figs. 8 and 9).

In another aspect, the present invention provides a concentrated culture liquid prepared by a method for producing a culture medium containing a growth factor or cytokine, which comprises culturing adipose stem cells on a scaffold and culturing the same three-dimensionally.

In another aspect, the present invention provides a composition for bone tissue regeneration comprising the concentrate as an active ingredient.

The term "osseous tissue" of the present invention is a term referring to a tissue that forms a bone. The bone is made up of fine-grained bone, and both ends of the central or iliac bone are associated with a spongy bone like a net. Most of the bones first develop as cartilage in the connective tissue and later turn into bone tissue, some of which are produced directly in the connective tissue. At the distal end, there is a joint surface in contact with the neighboring bone, and its surface is covered with articular cartilage, which is a supernatural bone. The sponge in the middle of the spongy is characterized by a constant arrangement. The osteocytes are arranged between the stratum corneum and are connected to other adjacent osteoclasts by protruding protrusions with irregular star shape. There is a rigid connective tissue periosteum on the surface of the bones, and nerve and blood vessels are distributed to protect and nourish the bones. If the periosteum is deficient, it becomes difficult to survive, start, regenerate, and the like of the bone. Bone content is 20% moisture, 35% organic and 35% inorganic, because the bone has a constant elasticity of organic matter. As age increases, minerals (mainly calcium phosphate) increase and bone hardness increases.

The term "regeneration" of the present invention generally refers to an action in which, when an organism loses a part of its body or its function, the tissue or organ of the part is regenerated and restored or restored to its original state. This regeneration ability is stronger as the system is simple and systematic and the degree of evolution is low. In one embodiment, the present invention provides the use of a culture medium in which 1% or 5% of a concentrated culture medium of the present invention is mixed with a culture medium for inducing differentiation of normal osteoclasts to a higher ALP activity (Fig. 8) and calcification 9), confirming that the culture solution obtained from the three-dimensional culture of the present invention efficiently regenerates the bone tissue, and confirmed that it can be used as a composition for regenerating bone tissue.

According to another aspect of the present invention, there is provided a method for culturing a stem cell, comprising culturing a culture medium comprising a growth factor or cytokine comprising a step of adhering adult stem cells to a scaffold and culturing the same three-dimensionally; And a step of loading the prepared culture liquid into a scaffold for grafting, wherein the scaffold is loaded with a culture medium.

The term "transplantation" of the present invention generally refers to the process of transferring the donor's cells, tissues or organs to the damaged tissue or organ of the recipient. Depending on the donor type, autografts transplanted their own cells or tissues elsewhere, allografts (eg, from humans to humans) that transplant cells, tissues, organs from a donor that is the same species as the recipient Can be classified as xenografts that transplant organs from a species (e.g., organs of an animal to humans). The subject of transplantation includes, without limitation, any cell, tissue or organ of the donor, as well as any material that can aid the regeneration of the injured tissue of the recipient. The transplantation can be performed by a method known in the art, and is not limited to the embodiment of the present invention. For example, surgery can be performed with surgery, and materials such as cells can be injected directly into the affected area.

The term "scaffold for implantation" of the present invention is a structure that is implanted together with a material to be implanted, and serves to support the material to be implanted. Therefore, it should be a substance having biocompatibility and high cell or tissue engraftment rate. Preferably, the implantable scaffold may be a biodegradable material, more preferably a collagen.

The term "loading " of the present invention refers to the process of containing a therapeutically effective substance in the implantable scaffold. Any method that satisfies these functions can be used without limitation. In the present invention, a stem cell enrichment culture fluid effective for regenerating bone tissue is applied to a collagen scaffold used as a graft scaffold to be absorbed.

The term "transplant" of the present invention refers to a support that isolates a damaged site from the outside or contains implanted cells or secreted therapeutic material and allows the cell or therapeutic material to stay, it means. Such grafts include, without limitation, various materials used in the art such as biodegradable synthetic polymers and natural materials as supports for tissue engineering. Preferably, it may be collagen. The transplant of the present invention was confirmed in a specific example that a collagen scaffold containing stem cell enrichment broth does not contain non-magnetic cells and thus does not cause an immunological rejection reaction and does not cause an inflammatory reaction (FIG. 11). Therefore, even without administering a separate immunosuppressive agent or an anti-inflammatory agent to inhibit the immune rejection reaction or the inflammatory reaction that may occur when various grafts are transplanted into the body, the grafted graft is stable in vivo without inducing immunological rejection or inflammation . The transplant of the present invention is a collagen scaffold including a stem cell concentration culture medium, and may further include mesenchymal stem cells in addition to the collagen scaffold containing the concentrate.

As another embodiment, the present invention provides There is provided an implant for bone tissue regeneration comprising a scaffold loaded with the prepared culture solution.

A graft composed of a collagen scaffold containing a growth medium obtained by concentrating a growth medium containing a large amount of growth factors and cytokines obtained by three-dimensionally culturing the stem cells of the present invention on a solid support was transplanted into a rat two- (Fig. 10), promoted migration of stem cells (Fig. 11) without inflammatory reaction (Fig. 12) and showed higher aggregate bioactivity than the control cells transplanted with stem cells (Fig. 13 to 15).

The culture solution obtained by culturing the stem cells of the present invention on a solid scaffold and culturing the cells in a three-dimensional manner contains a large amount of growth factors and cytokines. The concentrated culture solution is loaded on a collagen scaffold to be transplanted into a bone defect site, Indicating the effect of bone regeneration. Therefore, the concentrated culture solution of the present invention does not contain cells, and thus can be utilized as a composition for regenerating aggregates without worrying about an immune rejection reaction or an inflammatory reaction.

FIG. 1 shows an electric stimulation device (middle) in an in vitro culture system for electric stimulation, an electric stimulation intensity control device (left) in an abnormal current stimulator, and an actual state (right) of loading collagen cultured in an electric stimulation device .
FIG. 2 is an electron microscope photograph for confirming cell adhesion to a scaffold. FIG. From the left, there is a collagen scaffold with no cells attached, a scaffold attached with cells, and an experimental group (EC) with cells attached and electrical stimulation applied to the scaffold.
FIG. 3 is a diagram showing gene expression amounts of growth factors and cytokines through real-time RT-PCR. 2D and 3D cultures, respectively.
FIG. 4 is a graph showing the number of days of culturing by comparing the amount of gene expression according to the presence or absence of electric stimulation in a three-dimensional culture.
FIG. 5 is a graph showing the results of an enzyme-linked immunosorbent assay (ELISA) for comparing the expression levels of growth factors and cytokines according to the culture method. The expression levels of VEGF (vascular endothelial growth factor), bFGF (basic fibroblast growth factor) and IL-8 (interleukin-8) were measured and the expression levels of 2D and 3D cultures, Were measured and compared.
6 is a diagram showing a protein concentration filter used for concentration of a culture liquid.
7 is a graph showing the results of MTT analysis for confirming the cytotoxicity of the concentrated culture medium. Cell viability was measured for cells cultured for 3 days in a general culture medium in which the concentrate of the present invention was mixed at 1, 2.5 and 5%.
FIG. 8 shows ALP (alkaline phosphatase) activity according to culture conditions. FIG. A concentrated culture solution prepared by culturing stem cells in the presence or absence of electric stimulation in conditions of differentiation into bone cells (dex (dex) and dexamethasone) for differentiating into a control group (con (ascorbic acid,? -Glycerophosphate) ALP activity was measured in the experimental group added at the indicated concentration and the effect on osteoclast differentiation was confirmed.
FIG. 9 is a graph showing the result of staining of alizarin red S for confirming the influence of a concentrated culture solution on bone cell differentiation and the amount of alizarin red S obtained by decolorizing the same. Stem cells cultured in each condition stained with alizarin red S were photographed, decolorized with decolorizing solution, and absorbance of the solution was measured at a wavelength of 595 nm and converted to a concentration.
FIG. 10 is a diagram showing the preparation of an experimental animal model with two open-mouth injuries and an animal experimental procedure using the same.
11 is a diagram showing the result of H & E staining for confirming the inflammatory reaction by the concentrated culture liquid.
FIG. 12 is a graph showing the results of stimulation of stem cell migration of a concentrated culture medium. FIG. The collagen scaffold loaded with the culture medium of the present invention and the culture medium of the present invention was inserted into each of the two lesion injured experimental animal models, and human embryo-derived stem cells labeled with a fluorescent dye, a human bone marrow-derived stem cell, were injected. As a comparative experimental group, a scaffold loaded with SDF-1 (stromal cell-derived factor-1), which promotes stem cell migration, was used.
13 is a graph showing the result of quantitative analysis of the degree of bone formation by micro CT imaging. A control cell loaded with stem cells into a scaffold, an experimental group (media-C) implanted with a scaffold loaded with a concentrate prepared by 3D culture, and a concentrate prepared by 3D culture with electrical stimulation The bone volume (BV), trabecular number (Tb.N), trabecular separation (Tb.Sp), and trabecular separation (Tb.Sp) of the loaded scaffold transplantation group (media- And trabecular thickness (Tb.Th) were measured.
14 is a view showing an image of a new bone tissue measured using a micro CT. Two implanted animals were implanted with each implant and four weeks later, micro CT was performed to image the newly formed bone tissue.
15 is a diagram showing the results of histological staining for confirming the degree of bone formation. The bone grafts were transplanted into the osseous animal model that caused 5 mm diameter bone defects in both openings and 4 weeks after transplantation, the degree of osteogenesis was evaluated histologically using Marsh Tri-color staining.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

Example  1: human Bone marrow origin Intermediate lobe Adult stem cells  Isolation and Culture

The human bone marrow as a source of adult stem cells of the present invention was obtained from a iliac bone aspirated puncture device with the consent of the patient and obtained permission from the university's institutional review board (IRB). The bone marrow fluid was extracted from the iliac crest region of the bone marrow donor by the method described in Vilamitjana-Amedee et al. (In Vitro Cell Dev. Biol. Anim., 1993, 29A (9): 699-707). Ficoll-Paque ^™ PLUS was added to separate the layers according to their density, and then the nucleated cell layer formed between the supernatant and the lower blood layer was separated. Separated nucleated cells were cultured in DMEM (Dulbecco's modified Eagle's medium) medium at a density of 3 to 4 × 10 ⁴ cells / cm ² under medium containing 10% fetal bovine serum (FBS) After 4 days, the cells were replaced with fresh medium, and a colony was formed. The cells were grown to 60 to 70% on a culture plate, and subculture was started. In order to obtain a sufficient number of cells, the number of subcultures was increased and experiments were carried out in the second subculture, and the remaining cells were stored frozen.

Example  2: Electrical stimulation and flat or stellate cell culture conditions

As a method for promoting the secretion of growth factors and cytokines during the culture of the isolated stem cells, the cells were cultured in three dimensions by attaching to the culture and / or stereoscopic scaffold while giving electrical stimulation. An apparatus designed to give electrical stimulation during culture is shown in Fig. As the electrode, a plate deposited with gold was used, and cells were allowed to adhere to the channel electrode. Electrical stimulation was applied in combination with stereogenic conditions. 2D plane cultures were cultured on a common Teflon culture dish while 3D culture was carried out by introducing a collagen scaffold and incubating the cells on the 2D collagen scaffold. The electrical stimulation was given differently depending on the culture conditions. In the 2D plane culture, seeding was carried out at a cell density of 1.7 × 10 ³ cells / cm ² and stimulation was carried out for 1.5 days at 1.5 μA, 250 μs, And attached to a collagen scaffold. At the time of 3D culture, 5 × 10 ⁵ cells were seeded into 5-mm collagen in a volume of 20 μl, allowed to adhere for 3 to 4 hours, added with the culture solution, 20 μA, 250 μs, and 100 Hz for 4 days. On the surface of the collagen scaffold used for 3D stereoculture, the experimental group to which human bone marrow-derived adult stem cells were adhered, and the cell type of the experimental group to which adult stem cells were attached to the collagen scaffold were injected on the fourth day of culture And confirmed by scanning electron microscopy.

As a result of the scanning electron microscopic observation, it was confirmed that the surface of the collagen scaffold had a porous surface capable of entering into the cell while proliferating, and the stem cell of the present invention was easily attached to the porous collagen scaffold, And it was confirmed that the cells were stably attached and cultured and proliferated even when electric stimulation was applied (Fig. 2).

Example  3: Real time Reverse transcriptase - Polymerase chain reaction real time reverse  전사화ASE-Polymerase chain reaction ; real time RT - PCR ) Gene expression analysis

First, the amount of gene expression was compared with that of steric cell culture. Real time RT-PCR was performed by a known method.

As a result, as shown in FIG. 3, it was confirmed that gene expression was significantly increased in 3D culture incubated with collagen scaffold than in 2D plane culture. Specifically, in 3D cultures, insulin-like growth factor-1 (IGF-1), BMP2 (bone morphogenetic protein 2) and CXCL2 (chemokine CXC motif ligand 2) 8 (interleukin-8) and VEGF (vascular endothelial growth factor) increased 1.2-fold (FIG. 3).

Furthermore, in order to confirm the effect of electric stimulation on gene expression in 3D culture, gene expression levels were measured on the 2nd and 4th days of 3D culture in the absence and presence of electrical stimulation, and the results are shown in FIG.

As a result, it was confirmed that gene expression of bone regeneration related factors was increased by electric stimulation although there was a difference in degree and timing. BMP2, VEGF, TGF-β, and bFGF genes increased 4.2 and 2.3 times (data not shown), 2.3 and 2.5 times on day 4, respectively, while the IGF-1 gene increased 1.6 times on the second day of culture. On the other hand, the expression of CXCL2 and IL-8 genes, which are cytokines contributing to stem cell and early immune response related cell migration, increased to 2.8-fold and 2.5-fold on day 4, respectively (FIG.

Example  4: ELISA ( enzyme - linked immunosorbent assay Analysis of protein expression using

It was confirmed from the above Example 3 that the amount of expression of osteogenic regeneration-related growth factors, stem cells, and early immune response-related cell migration-promoting cytokine genes was increased when cultured under 3D culture and / or electrical stimulation. The expression levels of the proteins were compared using an ELISA.

Specifically, the cells were cultured for 3 days and the culture broth was collected. The vascular endothelial growth factor (VEGF) required for osteogenesis in the culture medium, bFGF (basic fibroblast growth factor), which plays an important role in cell proliferation and development, The expression level of IL-8 (interleukin-8), a cytokine involved in stem cell migration, was confirmed.

As a result, VEGF, which plays an important role in angiogenesis, was present at a concentration 19 times higher in 3D culture using collagen scaffold than in 2D plane culturing. When cultured in 2D and 3D, electric stimulation (EC) Stimulation increased 1.37 and 1.13 fold, respectively. On the other hand, bFGF was present at a concentration about 2 times higher in 3D culture than 2D culture, whereas 2D culture showed electrical stimulation but not 3D culture (data not shown). Finally, cytokine IL-8 was found to be secreted in 26.5-fold higher concentration than in 2D plane culture only by 3D stereoculture, and increased by 1.38-fold and 2.02-fold by electrical stimulation, respectively (FIG. 5).

In conclusion, a culture medium containing high concentration of growth factors and cytokines (IGF-1, VEGF, BMP2, bFGF, CXCL2, IL-8) could be obtained by 3D stereoculture using collagen scaffold, , And the culture supernatant of VEGF, bFGF and IL-8 was increased.

Example  5: Enrichment of stem cell culture and evaluation of activity of concentrated culture

Stem cells were cultured by the method of Example 2, and the resulting culture was concentrated using a protein concentration filter (sartorius, vivaspin 2). Using this filter, 1.5 ml of the culture solution can be highly concentrated at 30 μl. Table 1 summarizes the total volume of the culture medium in the 2D and 3D cultures and the time required to concentrate them using the column.

As a result, when 3D culture conditions in which cells are attached to collagen scaffold are used, only 3 ml of culture medium is used for 1 × 10 ⁶ cell cultures, while when cultured in a plane (2D) on a culture dish, 14 ml of culture medium was required to cultivate the cells, and thus the concentration time was also 3.5 times or more. These results support that the 3D culture method of the present invention can concentrate the culture medium in a small amount of medium and in a short time.

It was confirmed through Examples 3 and 4 that the growth factor and cytokine were present at a higher concentration in the culture solution obtained by 3D culture than the culture solution obtained from 2D culture, The concentration of the culture solution using the protein concentration filter was performed only for the 3D culture solution.

Table 2 lists the concentration of protein measured before and after filtration and after filtration to confirm the concentration. However, since the concentration of protein before filtration and the concentration of protein concentrated in the filter after filtration are maintained, growth factors and cytokines having increased expression in most of the cultures are not lost to the outside of the filter Was successfully completed.

The concentrated culture was kept in a freezer and then thawed according to necessity in the experiment.

Example  6: Influence of stem cell concentration on cell proliferation

In order to confirm the cytotoxicity and the effect on the cell proliferation of the concentrate culture medium, the culture obtained by culturing with the control (con) and electric stimulation (EC) was concentrated and mixed with the culture medium at the ratios of 1, 2.5 and 5% For 3 days and confirmed by using cell counting kit-8 (CCK-8).

As shown in FIG. 7, the experimental groups cultured with the concentrated culture medium mixture showed increased absorbance than the control group, indicating that the cytotoxicity due to the concentrated culture medium was not observed. Furthermore, 1.17-fold and 1.23-fold higher cell proliferation was observed in the experimental group to which the 1% and 2.5% concentrated cultures were added, respectively. On the other hand, the addition of the concentrate of the culture obtained by the electric stimulation gave similar effects on cell proliferation.

Example  7: Effect of Stem Cell Concentration Culture on Bone Differentiation

ALP (alkaline phosphatase) activity and alizarin red S stain were examined to confirm the effect of the stem cell enrichment culture of the present invention on osteoblast differentiation of stem cells. ALP is present in many parts of the body, especially bone and liver. It exists in osteoblast, which is an osteoblast, and Alizarin Red S binds to metal ion, so calcium deposits in the body, mineralization sites. Therefore, the level of bone formation can be determined by the degree of ALP activity and alizarin red S staining.

First, stem cells were treated with control (con) and bone cell differentiation conditions (dex). The control group was cultured in a medium supplemented with ascorbic acid and? -Glycerophosphate, and dexametasone was added to the control medium as a positive control group for bone cell differentiation. The experimental group was cultured in a medium containing 1% or 5% of the culture obtained by 3D culture under the electric stimulation zone / absence.

FIG. 8 shows the results of ALP activity measurement of the control and experimental groups. As a result, the ability to differentiate into osteocytes was increased by 16 to 37% when a culture medium containing 1% or 5% of the concentrate culture medium was used, compared to osteocyte differentiation conditions in which only dexamethasone was added to the control medium. However, no further increase due to electrical stimulation was observed.

Fig. 9 shows the results of alizarin red S staining. First, using a culture medium (3D-con 1%, 3D-EC 1%, 3D-con 5% and 3D-EC) supplemented with a positive control group (Dex) and a concentrated culture medium except for the negative control 5%). After 21 days, cells were fixed with 10% formalin, and fixed solution was removed with distilled water. Then, the alizarin red S staining solution was added and reacted for 5 to 10 minutes. Then, the staining solution was removed with distilled water and left in PBS for 15 minutes. Stem cells differentiated into stained bone cells were photographed and examined for their degree of differentiation into osteocytes by visual observation, and then decolorization was carried out to quantify them. After staining with a de-stain solution for 15 minutes, the resulting solution was subjected to ELISA at 595 nm to quantitatively compare the degree of alizarin red S staining. The concentrations of stained alizarin red S, quantified and expressed as numerical values, were in agreement with the results of the previous visual evaluation. In other words, in the experimental group (3D-con 1%) in which the positive control (Dex) and the concentrated culture obtained by 3D culture without electrical stimulation were differentiated into the culture medium containing 1%, the degree of differentiation into osteocytes was small, (3D-EC 1%, 3D-EC 5%) in which 1% or 5% of the concentrated culture solution obtained by differentiating the culture with the culture solution containing 5% (3D-con 5%) showed differentiation into bone cells.

Example  8: Animal Experimental Model Osteogenesis  Activity evaluation

The concentrated, frozen culture was thawed and transplanted into two open-cell defect models in rats to confirm the degree of osteogenesis activity in vivo. Two open cuts with a diameter of 5 mm were formed by using a trephine bar on the right and left sides of the skull of rats, and then adult stem cells, a culture medium obtained by 3D culture (media-C) A medium (EC) was loaded onto the collagen scaffold, which was a biodegradable solid support, and transplanted into the injured area, and lysed at 2 weeks and 4 weeks to confirm the degree of bone regeneration. FIG. 10 shows the process of preparing the two open defect models and the implantation process of the implanted material.

Example  9: Confirmation of induction of inflammatory reaction in the concentrate

To determine whether the inflammatory response was induced by the transplanted concentrate, the transplanted rats were sacrificed 2 weeks after transplantation, and tissue sections were prepared and H & E staining was used to identify inflammatory cells.

As a result, as shown in Fig. 11, no cells that seemed to be inflammatory cells in the stem cell transplantation group and the Media-Con and Media-EC transplantation groups were found. In other words, the inflammatory reaction caused by the transplanted stem cells and the concentrated culture solution was not induced. However, it has been confirmed that some of the transplanted collagen still remains.

Example  10: Stimulation of stem cell migration in concentrated culture medium

In the same manner as in Example 9, two defects were made in the rat skull, a scaffold loaded with a culture medium on one side and a scaffold loaded with a concentrated culture obtained by 3D culture on the other side were transplanted. On the day following the transplantation, 5 × 10 ⁶ human bone marrow-derived mesenchymal stem cells (hBMSCs) labeled with a fluorescent dye, a fluorescent dye, were injected through the tail vein of the rat. The positive control group was prepared by using SDF-1 (50 μg / ml), which is known as a stem cell migration promoting substance, instead of the concentrate in the same experimental animal model. To confirm the migration of stem cells, the rat was sacrificed and the scaffold was recovered and frozen and cut with a confocal microscope.

As a result, as shown in Fig. 12, fluorescence of human bone marrow-derived stem cells injected from the tail was not observed in the control group injected with the general culture medium, but it was observed in the positive control group and the experimental group. In conclusion, it was confirmed that the enriched culture medium of stem cells contains a large amount of substances having the effect of promoting stem cell migration, and thus promotes stem cell migration similar to that when SDF-1, a stem cell migration promoting substance, is injected, It is shown that only transplantation of the culture medium without transplantation of stem cells capable of differentiating into tissues can induce tissue regeneration by promoting migration of autologous stem cells in vivo to the wound site.

Example  11: Bone regeneration  effect

<11-1> Micro CT Using Osteogenesis  Quantitative analysis

The degree of osteogenesis from the scaffold loaded with the transplanted stem cells or the concentrate was quantitatively analyzed through 3-D micro-CT (FIG. 13). As a control group, stem cells were attached to a 3D collagen scaffold and cultured for 4 days. In the experimental group (Media-C and Media-EC), the concentrate was loaded and loaded in the same scaffold without cells, which showed 219 and 277% increase in bone volume (BV) . The number of trabecular bone was also increased by 202 and 270%, but the distribution and thickness of trabecular bone formed were similar in all groups. The synergistic effect of the electrical stimulation was not observed in the concentrated culture obtained by the electric stimulation. However, regardless of the electric stimulation, the experimental group transplanted with the concentrated culture obtained by 3D culture showed better bone regeneration effect than the control group transplanted with stem cells Respectively. Fig. 14 shows a three-dimensional image obtained by micro CT imaging. Imagery of newly formed bone (yellow) 4 weeks after loading stem cell or enriched culture fluid into bone defect model was made. The new bone tissues (yellow) were observed more frequently in the experimental group (Media-C and Media-EC) than in the stem cell transplantation (Cell), which was consistent with the results of the previous micro-CT analysis.

<11-2> Using histological staining Osteogenesis  evaluation

Histological examination was performed using Masson trichrome stain for the analysis of new bone tissue from scaffolds loaded with stem cells or concentrate. Scaffolds loaded with stem cells or enriched cultures were transplanted into two open-cell defect models prepared by the method described in Example 8, and stained by dying after 4 weeks. As shown in FIG. 15, in the new bone tissue, in the experimental group (Media-C and Media-EC) in which the enrichment culture fluid was transplanted from the control cell (cell) transplanted with the stem cells in accordance with the micro-CT quantitative analysis result and the three- More was observed. Specifically, in the group transplanted with stem cells, only a collagen scaffold was observed in which new bone tissues were hardly recognized and were transplanted together with a solid support. On the other hand, a significant amount of new bone tissue was observed in the case of transplantation of the concentrate culture fluid, and the collagen scaffold was degraded and decreased in proportion thereto.

Claims (18)

delete delete delete delete delete delete delete delete delete (Insulin-like growth factor-1), TGF-beta 1 (transforming growth factor-beta 1), and basic fibroblast growth factor (bFGF), which are prepared by culturing bone marrow-derived mesenchymal stem cells on a scaffold, ), A growth factor selected from the group consisting of bone morphogenetic protein 2 (BMP2) or vascular endothelial growth factor (VEGF), or a cytokine such as CXCL2 (chemokine CXC motif ligand 2) or IL-8 (interleukin-8) A composition for regenerating bone tissue comprising a culture medium as an active ingredient.
(a) TGF-beta1 (transforming growth factor-beta1), bFGF (basic), and IGF-1 (insulin-like growth factor-1) prepared by culturing bone marrow-derived mesenchymal stem cells on a scaffold, a cytokine such as CXCL2 (chemokine CXC motif ligand 2) or IL-8 (interleukin-8), which is selected from the group consisting of fibroblast growth factor, bone morphogenetic protein 2 (BMP2) or vascular endothelial growth factor Preparing a culture medium containing the culture medium; And
(b) loading the prepared culture into a grafting scaffold.
A method for producing an implant for regenerating bone tissue, comprising a scaffold for implantation in which a culture medium is loaded.
12. The method of claim 11,
Wherein the implantable scaffold is a biodegradable material.
13. The method of claim 12,
Wherein the biodegradable material is collagen.
A bone regeneration implant for bone grafts comprising a scaffold for implantation loaded with a culture prepared by the method of claim 11.
11. The composition for bone regeneration according to claim 10, wherein the culturing further comprises culturing bone marrow-derived mesenchymal stem cells with electrical stimulation.
11. The composition of claim 10, wherein the scaffold is a collagen scaffold.
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